Crispr effector system based diagnostics

ABSTRACT

The embodiments disclosed herein utilized RNA targeting effectors to provide a robust CRISPR-based diagnostic with attomolar sensitivity. Embodiments disclosed herein can detect broth DNA and RNA with comparable levels of sensitivity and can differentiate targets from non-targets based on single base pair differences. Moreover, the embodiments disclosed herein can be prepared in freeze-dried format for convenient distribution and point-of-care (POC) applications. Such embodiments are useful in multiple scenarios in human health including, for example, viral detection, bacterial strain typing, sensitive genotyping, and detection of disease-associated cell free DNA.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.15/917,546 filed Mar. 9, 2018, which is a continuation of InternationalPatent Application No. PCT/US2017/065477 filed Dec. 8, 2017, whichclaims the benefit of U.S. Provisional Application No. 62/432,553 filedDec. 9, 2016, U.S. Provisional Application No. 62/456,645 filed Feb. 8,2017, U.S. Provisional Application No. 62/471,930 filed Mar. 15, 2017,U.S. Provisional Application No. 62/484,869 filed Apr. 12, 2017, andU.S. Provisional Application No. 62/568,268 filed Oct. 4, 2017. Theentire contents of the above-identified applications are hereby fullyincorporated herein by reference.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH

This invention was made with government support under grant numbersMH100706 and MH110049 granted by the National Institutes of Health, andgrant number HDTRA1-14-1-0006 granted by the Defense Threat ReductionAgency. The government has certain rights in the invention.

TECHNICAL FIELD

The subject matter disclosed herein is generally directed to rapiddiagnostics related to the use of CRISPR effector systems.

BACKGROUND

Nucleic acids are a universal signature of biological information. Theability to rapidly detect nucleic acids with high sensitivity andsingle-base specificity on a portable platform has the potential torevolutionize diagnosis and monitoring for many diseases, providevaluable epidemiological information, and serve as a generalizablescientific tool. Although many methods have been developed for detectingnucleic acids (Du et al., 2017; Green et al., 2014; Kumar et al., 2014;Pardee et al., 2014; Pardee et al., 2016; Urdea et al., 2006), theyinevitably suffer from trade-offs among sensitivity, specificity,simplicity, and speed. For example, qPCR approaches are sensitive butare expensive and rely on complex instrumentation, limiting usability tohighly trained operators in laboratory settings. Other approaches, suchas new methods combining isothermal nucleic acid amplification withportable platforms (Du et al., 2017; Pardee et al., 2016), offer highdetection specificity in a point-of-care (POC) setting, but havesomewhat limited applications due to low sensitivity. As nucleic aciddiagnostics become increasingly relevant for a variety of healthcareapplications, detection technologies that provide high specificity andsensitivity at low cost would be of great utility in both clinical andbasic research settings.

SUMMARY

In one aspect, the invention provides a nucleic acid detection systemcomprising: a CRISPR system comprising an effector protein and one ormore guide RNAs designed to bind to corresponding target molecules; anRNA-based masking construct; and optionally, nucleic acid amplificationreagents to amplify target RNA molecules in a sample. In another aspect,the embodiments provide a polypeptide detection system comprising: aCRISPR system comprising an effector protein and one or more guide RNAsdesigned to bind a trigger RNA, an RNA-based masking construct; and oneor more detection aptamers comprising a masked RNA polymerase promoterbinding site or a masked primer binding site.

In further embodiments, the system may further comprise nucleic acidamplification reagents. The nucleic acid amplification reagents maycomprise a primer comprising an RNA polymerase promoter. In certainembodiments, sample nucleic acids are amplified to obtain a DNA templatecomprising an RNA polymerase promoter, whereby a target RNA molecule maybe generated by transcription. The nucleic acid may be DNA and amplifiedby any method described herein. The nucleic acid may be RNA andamplified by a reverse transcription method as described herein. Theaptamer sequence may be amplified upon unmasking of the primer bindingsite, whereby a trigger RNA is transcribed from the amplified DNAproduct. The target molecule may be a target DNA and the system mayfurther comprise a primer that binds the target DNA and comprises an RNApolymerase promoter.

In one example embodiment, the CRISPR system effector protein is anRNA-targeting effector protein. Example RNA-targeting effector proteinsinclude Cas13b and C2c2 (now known as Cas13a). It will be understoodthat the term “C2c2” herein is used interchangeably with “Cas13a”. Inanother example embodiment, the RNA-targeting effector protein is C2c2.In other embodiments, the C2c2 effector protein is from an organism of agenus selected from the group consisting of: Leptotrichia, Listeria,Corynebacter, Sutterella, Legionella, Treponema, Filifactor,Eubacterium, Streptococcus, Lactobacillus, Mycoplasma, Bacteroides,Flaviivola, Flavobacterium, Sphaerochaeta, Azospirillum,Gluconacetobacter, Neisseria, Roseburia, Parvibaculum, Staphylococcus,Nitratifractor, Mycoplasma, Campylobacter, and Lachnospira, or the C2c2effector protein is an organism selected from the group consisting of:Leptotrichia shahii, Leptotrichia. wadei, Listeria seeligeri,Clostridium aminophilum, Carnobacterium gallinarum, Paludibacterpropionicigenes, Listeria weihenstephanensis, or the C2c2 effectorprotein is a L. wadei F0279 or L. wadei F0279 (Lw2) C2C2 effectorprotein. In another embodiment, the one or more guide RNAs are designedto detect a single nucleotide polymorphism, splice variant of atranscript, or a frameshift mutation in a target RNA or DNA.

In other embodiments, the one or more guide RNAs are designed to bind toone or more target molecules that are diagnostic for a disease state. Instill further embodiments, the disease state is an infection, an organdisease, a blood disease, an immune system disease, a cancer, a brainand nervous system disease, an endocrine disease, a pregnancy orchildbirth-related disease, an inherited disease, or anenvironmentally-acquired disease. In still further embodiments, thedisease state is cancer or an autoimmune disease or an infection.

In further embodiments, the one or more guide RNAs are designed to bindto one or more target molecules comprising cancer specific somaticmutations. The cancer specific mutation may confer drug resistance. Thedrug resistance mutation may be induced by treatment with ibrutinib,erlotinib, imatinib, gefitinib, crizotinib, trastuzumab, vemurafenib,RAF/MEK, check point blockade therapy, or antiestrogen therapy. Thecancer specific mutations may be present in one or more genes encoding aprotein selected from the group consisting of Programmed Death-Ligand 1(PD-L1), androgen receptor (AR), Bruton's Tyrosine Kinase (BTK),Epidermal Growth Factor Receptor (EGFR), BCR-Abl, c-kit, PIK3CA, HER2,EML4-ALK, KRAS, ALK, ROS1, AKT1, BRAF, MEK1, MEK2, NRAS, RAC1, and ESR1.The cancer specific mutation may be a mutation in a gene selected fromthe group consisting of CASP8, B2M, PIK3CA, SMC1A, ARID5B, TET2, ALPK2,COL5A1, TP53, DNER, NCOR1, MORC4, CIC, IRF6, MYOCD, ANKLE1, CNKSR1, NF1,SOS1, ARID2, CUL4B, DDX3X, FUBP1, TCP11L2, HLA-A, B or C, CSNK2A1, MET,ASXL1, PD-L1, PD-L2, IDO1, IDO2, ALOX12B and ALOX15B, or copy numbergain, excluding whole-chromosome events, impacting any of the followingchromosomal bands: 6q16.1-q21, 6q22.31-q24.1, 6q25.1-q26, 7p11.2-q11.1,8p23.1, 8p11.23-p11.21 (containing IDO1, IDO2), 9p24.2-p23 (containingPDL1, PDL2), 10p15.3, 10p15.1-p13, 11p14.1, 12p13.32-p13.2, 17p13.1(containing ALOX12B, ALOX15B), and 22q11.1-q11.21.

In further embodiments, the one or more guide RNAs may be designed tobind to one or more target molecules comprising loss-of-heterozygosity(LOH) markers.

In further embodiments, the one or more guide RNAs may be designed tobind to one or more target molecules comprising single nucleotidepolymorphisms (SNP). The disease may be heart disease and the targetmolecules may be VKORC1, CYP2C9, and CYP2C19.

In further embodiments, the disease state may be a pregnancy orchildbirth-related disease or an inherited disease. The sample may be ablood sample or mucous sample. The disease may be selected from thegroup consisting of Trisomy 13, Trisomy 16, Trisomy 18, Klinefeltersyndrome (47, XXY), (47, XYY) and (47, XXX), Turner syndrome, Downsyndrome (Trisomy 21), Cystic Fibrosis, Huntington's Disease, BetaThalassaemia, Myotonic Dystrophy, Sickle Cell Anemia, Porphyria,Fragile-X-Syndrome, Robertsonian translocation, Angelman syndrome,DiGeorge syndrome and Wolf-Hirschhorn Syndrome.

In further embodiments, the infection is caused by a virus, a bacterium,or a fungus, or the infection is a viral infection. In specificembodiments, the viral infection is caused by a double-stranded RNAvirus, a positive sense RNA virus, a negative sense RNA virus, aretrovirus, or a combination thereof, or the viral infection is causedby a Coronaviridae virus, a Picornaviridae virus, a Caliciviridae virus,a Flaviviridae virus, a Togaviridae virus, a Bornaviridae, aFiloviridae, a Paramyxoviridae, a Pneumoviridae, a Rhabdoviridae, anArenaviridae, a Bunyaviridae, an Orthomyxoviridae, or a Deltavirus, orthe viral infection is caused by Coronavirus, SARS, Poliovirus,Rhinovirus, Hepatitis A, Norwalk virus, Yellow fever virus, West Nilevirus, Hepatitis C virus, Dengue fever virus, Zika virus, Rubella virus,Ross River virus, Sindbis virus, Chikungunya virus, Borna disease virus,Ebola virus, Marburg virus, Measles virus, Mumps virus, Nipah virus,Hendra virus, Newcastle disease virus, Human respiratory syncytialvirus, Rabies virus, Lassa virus, Hantavirus, Crimean-Congo hemorrhagicfever virus, Influenza, or Hepatitis D virus.

In other embodiments of the invention, the RNA-based masking constructsuppresses generation of a detectable positive signal or the RNA-basedmasking construct suppresses generation of a detectable positive signalby masking the detectable positive signal, or generating a detectablenegative signal instead, or the RNA-based masking construct comprises asilencing RNA that suppresses generation of a gene product encoded by areporting construct, wherein the gene product generates the detectablepositive signal when expressed.

In further embodiments, the RNA-based masking construct is a ribozymethat generates the negative detectable signal, and wherein the positivedetectable signal is generated when the ribozyme is deactivated, or theribozyme converts a substrate to a first color and wherein the substrateconverts to a second color when the ribozyme is deactivated.

In other embodiments, the RNA-based masking agent is an RNA aptamer, orthe aptamer sequesters an enzyme, wherein the enzyme generates adetectable signal upon release from the aptamer by acting upon asubstrate, or the aptamer sequesters a pair of agents that when releasedfrom the aptamers combine to generate a detectable signal.

In another embodiment, the RNA-based masking construct comprises an RNAoligonucleotide to which a detectable ligand and a masking component areattached. In another embodiment, the detectable ligand is a fluorophoreand the masking component is a quencher molecule, or the reagents toamplify target RNA molecules such as, but not limited to, NASBA or RPAreagents.

In another aspect, the invention provides a diagnostic device comprisingone or more individual discrete volumes, each individual discrete volumecomprising a CRISPR effector protein, one or more guide RNAs designed tobind to corresponding target molecule, an RNA-based masking construct,and optionally further comprise nucleic acid amplification reagents.

In another aspect, the invention provides a diagnostic device comprisingone or more individual discrete volumes, each individual discrete volumecomprising a CRISPR effector protein, one or more guide RNAs designed tobind to a trigger RNA, one or more detection aptamers comprising amasked RNA polymerase promoter binding site or a masked primer bindingsite, and optionally further comprising nucleic acid amplificationreagents.

In some embodiments, the individual discrete volumes are droplets, orthe individual discrete volumes are defined on a solid substrate, or theindividual discrete volumes are microwells, or the individual discretevolumes are spots defined on a substrate, such as a paper substrate.

In one embodiment, the RNA targeting effector protein is a CRISPR TypeVI RNA-targeting effector protein such as C2c2 or Cas13b. In certainexample embodiments, the C2c2 effector protein is from an organismselected from the group consisting of: Leptotrichia, Listeria,Corynebacter, Sutterella, Legionella, Treponema, Filifactor,Eubacterium, Streptococcus, Lactobacillus, Mycoplasma, Bacteroides,Flaviivola, Flavobacterium, Sphaerochaeta, Azospirillum,Gluconacetobacter, Neisseria, Roseburia, Parvibaculum, Staphylococcus,Nitratifractor, Mycoplasma and Campylobacter, or the C2c2 effectorprotein is selected from the group consisting of: Leptotrichia shahii,L. wadei, Listeria seeligeri, Lachnospiraceae bacterium, Clostridiumaminophilum, Carnobacterium gallinarum, Paludibacter propionicigenes,Listeria weihenstephanensis, Listeriaceae bacterium, and Rhodobactercapsulatus, the C2c2 effector protein is a L. wadei F0279 or L. wadeiF0279 (Lw2) C2c2 effector protein. In another embodiment, the one ormore guide RNAs are designed to bind to one or more target RNA sequencesthat are diagnostic for a disease state.

In certain example embodiments, the RNA-based masking constructsuppresses generation of a detectable positive signal, or the RNA-basedmasking construct suppresses generation of a detectable positive signalby masking the detectable positive signal, or generating a detectablenegative signal instead, or the RNA-based masking construct comprises asilencing RNA that suppresses generation of a gene product encoded by areporting construct, wherein the gene product generates the detectablepositive signal when expressed.

In another example embodiment, the RNA-based masking construct is aribozyme that generates a negative detectable signal, and wherein thepositive detectable signal is generated when the ribozyme isdeactivated. In one example embodiment, the ribozyme converts asubstrate to a first color and wherein the substrate converts to asecond color when the ribozyme is deactivated. In another exampleembodiment, the RNA-based masking agent is an aptamer that sequesters anenzyme, wherein the enzyme generates a detectable signal upon releasefrom the aptamer by acting upon a substrate, or the aptamer sequesters apair of agents that when released from the aptamers combine to generatea detectable signal.

In another example embodiment, the RNA-based masking construct comprisesan RNA oligonucleotide to which are attached a detectable ligandoligonucleotide and a masking component. In certain example embodiments,the detectable ligand is a fluorophore and the masking component is aquencher molecule.

In another aspect, the invention provides a method for detecting targetRNAs in samples, comprising: distributing a sample or set of samplesinto one or more individual discrete volumes, the individual discretevolumes comprising a CRISPR system comprising an effector protein, oneor more guide RNAs, an RNA-based masking construct; incubating thesample or set of samples under conditions sufficient to allow binding ofthe one or more guide RNAs to one or more target molecules; activatingthe CRISPR effector protein via binding of the one or more guide RNAs tothe one or more target molecules, wherein activating the CRISPR effectorprotein results in modification of the RNA-based masking construct suchthat a detectable positive signal is produced; and detecting thedetectable positive signal, wherein detection of the detectable positivesignal indicates a presence of one or more target molecules in thesample.

In another aspect, the invention provides a method for detectingpeptides in samples, comprising: distributing a sample or set of samplesinto a set of individual discrete volumes, the individual discretevolumes comprising peptide detection aptamers, a CRISPR systemcomprising an effector protein, one or more guide RNAs, an RNA-basedmasking construct, wherein the peptide detection aptamers comprising amasked RNA polymerase site and configured to bind one or more targetmolecules; incubating the sample or set of samples under conditionssufficient to allow binding of the peptide detection aptamers to the oneor more target molecules, wherein binding of the aptamer to acorresponding target molecule exposes the RNA polymerase binding siteresulting in RNA synthesis of a trigger RNA; activating the CRISPReffector protein via binding of the one or more guide RNAs to thetrigger RNA, wherein activating the CRISPR effector protein results inmodification of the RNA-based masking construct such that a detectablepositive signal is produced; and detecting the detectable positivesignal, wherein detection of the detectable positive signal indicates apresence of one or more target molecules in a sample.

In certain example embodiments, such methods further comprise amplifyingthe sample RNA or the trigger RNA. In other embodiments, amplifying RNAcomprises amplification by NASBA or RPA.

In certain example embodiments, the CRISPR effector protein is a CRISPRType VI RNA-targeting effector protein, such as C2c2 or Cas13b. In otherexample embodiments, the C2c2 effector protein is from an organismselected from the group consisting of: Leptotrichia, Listeria,Corynebacter, Sutterella, Legionella, Treponema, Filifactor,Eubacterium, Streptococcus, Lactobacillus, Mycoplasma, Bacteroides,Flaviivola, Flavobacterium, Sphaerochaeta, Azospirillum,Gluconacetobacter, Neisseria, Roseburia, Parvibaculum, Staphylococcus,Nitratifractor, Mycoplasma and Campylobacter, or the C2c2 effectorprotein is selected from the group consisting of: Leptotrichia shahii,L. wadei, Listeria seeligeri, Lachnospiraceae bacterium, Clostridiumaminophilum, Carnobacterium gallinarum, Paludibacter propionicigenes,Listeria weihenstephanensis, Listeriaceae bacterium, and Rhodobactercapsulatus. In a specific embodiment, the C2c2 effector protein is a L.wadei F0279 or L. wadei F0279 (Lw2) C2C2 effector protein.

In certain example embodiments, the one or more guide RNAs are designedto bind to one or more target molecules that are diagnostic for adisease state. In certain other example embodiments, the disease stateis an infection, an organ disease, a blood disease, an immune systemdisease, a cancer, a brain and nervous system disease, an endocrinedisease, a pregnancy or childbirth-related disease, an inheriteddisease, or an environmentally-acquired disease, cancer, or a fungalinfection, a bacterial infection, a parasite infection, or a viralinfection.

In certain example embodiments, the RNA-based masking constructsuppresses generation of a detectable positive signal, or the RNA-basedmasking construct suppresses generation of a detectable positive signalby masking the detectable positive signal, or generating a detectablenegative signal instead, or the RNA-based masking construct comprises asilencing RNA that suppresses generation of a gene product encoded by areporting construct, wherein the gene product generates the detectablepositive signal when expressed, or the RNA-based masking construct is aribozyme that generates the negative detectable signal, and wherein thepositive detectable signal is generated when the ribozyme isinactivated. In other example embodiments, the ribozyme converts asubstrate to a first state and wherein the substrate converts to asecond state when the ribozyme is inactivated, or the RNA-based maskingagent is an aptamer, or the aptamer sequesters an enzyme, wherein theenzyme generates a detectable signal upon release from the aptamer byacting upon a substrate, or the aptamer sequesters a pair of agents thatwhen released from the aptamers combine to generate a detectable signal.In still further embodiments, the RNA-based masking construct comprisesan RNA oligonucleotide with a detectable ligand on a first end of theRNA oligonucleotide and a masking component on a second end of the RNAoligonucleotide, or the detectable ligand is a fluorophore and themasking component is a quencher molecule.

These and other aspects, objects, features, and advantages of theexample embodiments will become apparent to those having ordinary skillin the art upon consideration of the following detailed description ofillustrated example embodiments.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1—is a schematic of an example C2c2 based CRISPR effector system.

FIG. 2A—provides schematic of the CRISPR/C2c2 locus from Leptotrichiawadei. Representative crRNA structures from LwC2c2 and LshC2c2 systemsare shown. (SEQ. I.D. Nos. 142 and 143) FIG. 2B. Schematic of in vivobacterial assay for C2c2 activity. A protospacer is cloned upstream ofthe beta-lactamase gene in an ampicillin-resistance plasmid, and thisconstruct is transformed into E. coli expressing C2c2 in conjunctionwith either a targeting or non-targeting spacer. Successfultransformants are counted to quantify activity.

FIG. 2C. Quantitation of LwC2c2 and LshC2c2 in vivo activity. (n=2biological replicates; bars represent mean±s.e.m.) FIG. 2D. Final sizeexclusion gel filtration of LwC2c2. FIG. 2E. Coomassie blue stainedacrylamide gel of LwC2c2 stepwise purification. FIG. 2F. Activity ofLwC2c2 against different PFS targets. LwC2c2 was targeted againstfluorescent RNA with variable 3′ PFS flanking the spacer, and reactionproducts were visualized on denaturing gel. LwC2c2 shows a slightpreference against G PFS.

FIG. 3—Shows detection of an example masking construct at differentdilutions using 1 μg, 100 ng, 10 ng, and 1 ng of target with 4 differentamounts of protein/crRNA (1:4, 1:16, 1:32, 1:64) with 2 pools of crRNAs,no crRNA condition, technical duplicates, in (96+48)*2=288 reactions,measured in 5 min interval over 3 hours.

FIG. 4—Shows detection of an example masking construct at differentdilutions using 1 μg, 100 ng, 10 ng, and 1 ng of target with 4 differentamounts of protein/crRNA (1:4, 1:16, 1:32, 1:64) with 2 pools of crRNAs,no crRNA condition, technical duplicates, in (96+48)*2=288 reactions,measured in 5 min interval over 3 hours.

FIG. 5—Shows detection of an example masking construct at differentdilutions using 1 μg, 100 ng, 10 ng, and 1 ng of target with 4 differentamounts of protein/crRNA (1:4, 1:16, 1:32, 1:64) with 2 pools of crRNAs,no crRNA condition, technical duplicates, in (96+48)*2=288 reactions,measured in 5 min interval over 3 hours.

FIG. 6—Shows detection of an example masking construct at differentdilutions using 1 μg, 100 ng, 10 ng, and 1 ng of target with 4 differentamounts of protein/crRNA (1:4, 1:16, 1:32, 1:64) with 2 pools of crRNAs,no crRNA condition, technical duplicates, in (96+48)*2=288 reactions,measured in 5 min interval over 3 hours.

FIG. 7—provides a schematic of an example detection scheme using amasking construct and CRISPR effector protein, in accordance withcertain example embodiments.

FIG. 8—provides a set of graphs showing changes in fluorescence overtime when detecting a target using different pools of guide RNAs.

FIG. 9—provides a graph showing the normalized fluorescence detectedacross different dilutions of target RNA at varying concentrations ofCRISPR effector protein.

FIG. 10—is a schematic showing the general steps of a NASBAamplification reaction.

FIG. 11—provides a graph showing detection of nucleic acid target ssRNA1 amplified by NASBA with three different primer sets and then subjectedto C2c2 collateral detection using a quenched fluorescent probe. (n=2technical replicates; bars represent mean±s.e.m.)

FIG. 12—provides a graph showing that the collateral effect may be usedto detect the presence of a lentiviral target RNA.

FIG. 13—provides a graph demonstrating that the collateral effect andNASBA can detect species at aM concentrations.

FIG. 14—provides a graph demonstrating that the collateral effect andNASBA quickly discriminate low concentration samples.

FIG. 15—Shows that normalized fluorescence at particular time points ispredictive of sample input concentration. Fluorescence measurements fromCas13a detection without amplification are correlated with input RNAconcentration. (n=2 biological replicates; bars represent mean±s.e.m.).

FIG. 16—provides a schematic of the RPA reaction, showing theparticipating components in the reaction.

FIG. 17—schematic of SHERLOCK; provides a schematic showing detection ofboth DNA or RNA targets via incorporation of an RPA or an RT-RPA stepaccordingly. Upon recognition of target RNA, the collateral effectcauses C2c2 to cut the cleavage reporter, generating fluorescence.Single-molecule amounts of RNA or DNA can be amplified to DNA viarecombinase polymerase amplification (RPA) and transcribed to produceRNA, which is then detected by C2c2.

FIG. 18—provides a schematic of ssRNA target detected via the C2c2collateral detection (SEQ. I.D. Nos. 144 and 145).

FIG. 19—provides a set of graphs demonstrating single molecule DNAdetection using RPA (i.e. within 15 minutes of C2c2 addition).

FIG. 20—provides a set of graphs demonstrating that mixing T7 polymeraseinto a RPA reaction does adversely affect DNA detection.

FIG. 21—provides a set of graphs demonstrating that mixing polymeraseinto an RPA reaction does not adversely affect DNA detection.

FIG. 22—provides a graph demonstrating that RPA, T7 transcription, andC2c2 detection reactions are compatible and achieve single moleculedetection when incubated simultaneously (n=2 technical replicates; barsrepresent mean±s.e.m.).

FIG. 23—provides a set of graphs demonstrating the efficacy of quickRPA-RNA time incubations.

FIG. 24—provides a set of graphs demonstrating that increasing T7polymerase amount boosts sensitivity for RPA-RNA.

FIG. 25—provides a set of graphs showing results from an RPA-DNAdetection assay using a one-pot reaction with 1.5× enzymes. Singlemolecule (2 aM) detection achieved as early as 30 minutes.

FIG. 26—provides a set of graphs demonstrating that an RPA-DNA one-potreaction demonstrates a quantitative decrease in fluorescence relativeto input concentration. The fitted curve reveals relationship betweentarget input concentration and output fluorescence.

FIG. 27A, 27B—provides a set of graphs demonstrating that (FIG. 27A)C2c2 detection of RNA without amplification can detect ssRNA target atconcentrations down to 50 fM. (n=2 technical replicates; bars representmean±s.e.m.), and that (FIG. 27B) the RPA-C2c2 reaction is capable ofsingle-molecule DNA detection (n=4 technical replicates; bars representmean±s.e.m.).

FIG. 28—provides a set of graphs demonstrating that a C2c2 signalgenerated in accordance with certain example embodiments can detect a 20pM target on a paper substrate.

FIG. 29—provides a graph showing that a specific RNAse inhibitor iscapable of removing background signal on paper.

FIG. 30 is a set of graphs showing detection using systems in accordancewith certain example embodiments on glass fiber substrates.

FIGS. 31A-31D—provides a set of graphs providing (FIG. 31A) a schematicof Zika RNA detection in accordance with certain example embodiments.Lentivirus was packaged with Zika RNA or homologous Dengue RNA fragmentstargeted by C2c2 collateral detection. Media is harvested after 48 hoursand subjected to thermal lysis, RT-RPA, and C2c2 detection. FIG.31B—RT-RAP-C2c2 detection is capable of highly sensitive detection ofthe Zika lentiviral particles (n=4 technical replicates, two-tailedStudent t-test; *****, p<0.0001; bars represent mean±s.e.m.) FIG. 31Cshows a schematic of Zika RNA detection using freeze-dried C2c2 onpaper, in accordance with certain example embodiments. FIG. 31D showsthat a paper-based assay is capable of highly sensitive detection ofZika lentiviral particles (n-4 technical replicates, two-tailed Studentt-test; ****, p<0.0001; **, p<0.01, bars represent mean±s.e.m.).

FIG. 32A is a schematic for C2c2 detection of Zika RNA isolated fromhuman serum. Zika RNA in serum is subjected to reverse transcription,RNase H degradation of the RNA, RPA of the cDNA, and C2c2 detection.FIG. 32B is a graph showing that C2c2 is capable of highly sensitivedetection of human Zika serum samples. Concentrations of Zika RNA shownwere verified by qPCR (n=4 technical replicates, two-tailed Studentt-test; ****, p<0.0001; bars represent mean±s.e.m.).

FIGS. 33A-33G—provides a set of graphs demonstrating (FIG. 33A)freeze-dried C2c2 is capable of sensitive detection of ssRNA 1 in thelow femtomolar range. C2c2 is capable of rapid detection of a 200 pMssRNA 1 target on paper in liquid form (FIG. 33B) or freeze dried (FIG.33C). The reaction is capable of sensitive detection of synthesized ZikaRNA fragments in solution (FIG. 33D) (n=3) and in freeze-dried form(FIG. 33E) (n=3). FIG. 33F shows a quantitative curve for human zikacDNA detection showing significant correlation between inputconcentration and detected fluorescence. FIG. 33G is a graphillustrating C2c2 detection of ssRNA 1 performed in the presence ofvarying amounts of human serum (n=2 technical replicates, unlessotherwise noted; bars represent mean±s.e.m.).

FIG. 34A provides a schematic of C2c2 detection of 16S rRNA gene frombacterial genomes using a universal V3 RPA primer set. FIGS. 34B and 34Care graphs illustrating the ability to achieve sensitive and specificdetection of (FIG. 34B) E. coli or (FIG. 34C) P. aeruginosa gDNA usingan assay conducted in accordance with certain example embodiments (n=4technical replicates, two-tailed Student t-test; ****, p<0.0001; barsrepresent mean±s.e.m.). Ec, Escherichia coli; Kp, Klebsiella pneumoniae;Pa, Pseudomonas aeruginosa; Mt, Mycobacterium tuberculosis; Sa,Staphylococcus aureus.

FIG. 35A is a graph demonstrating detection of two differentcarbapenem-resistance genes (KPC and NDM-1) from four different clinicalisolates of Klebsiella pneumoniae. FIG. 35B is a graph demonstratingdetection of carbapenem-resistance genes (part A) is normalized as aratio of signal between the KPC and NDM-1 crRNA assays (n=2 technicalreplicates, two-tailed Student t-test; ****, p<0.0001; bars representmean±s.e.m.).

FIG. 36A illustrates that C2c2 is not sensitive to single mismatches,but can distinguish between single nucleotide differences in target whenloaded with crRNAs with additional mismatches. ssRNA targets 1-3 weredetected with 11 crRNAs, with 10 spacers containing synthetic mismatchesat various positions in the crRNA. Mismatched spacers did not showreduced cleavage of target 1, but showed inhibited cleavage of mismatchtargets 2 and 3 (SEQ. I.D. Nos. 146 through 159). FIG. 36B is aschematic showing the process for rational design of single-basespecific spacers with synthetic mismatches. Synthetic mismatches areplaced in proximity to the SNP or base of interest (SEQ. I.D. Nos. 160through 164). FIG. 36C is a graph showing that specific detection ofstrain SNPs allows for the differentiation of Zika African versusAmerican RNA targets differing by only one nucleotide using C2c2detection with truncated (23 nucleotide) crRNAs (n=2 technicalreplicates, one-tailed Student t-test; *, p<0.05; ****, p<0.0001; barsrepresent mean±s.e.m.).

FIG. 37A shows schematics of Zika virus African strain and Zika virusAmerican strain target regions and the crRNA sequences used fordetection (SEQ. I.D. Nos. 165 through 170). SNPs in the target arehighlighted red or blue and synthetic mismatches in the guide sequenceare colored red. FIG. 37B is a graph showing that highly specificdetection of strain SNPs allows for the differentiation of Zika Africanversus American RNA targets using SHERLOCK (n=2 technical replicates,two-tailed Student t-test; ****, p<0.0001; bars represent mean±s.e.m.)(SEQ. I.D. Nos. 171 through 176). FIG. 37C shows schematics of Denguestrain 3 and Dengue strain 1 target regions and the crRNA sequences usedfor detection. SNPs in the target are highlighted red or blue andsynthetic mismatches in the guide sequence are colored red. FIG. 37D isa graph showing that highly specific detection of strain SNPs allows forthe differentiation of Dengue strain 1 versus strain 3 RNA targets usingSHERLOCK (n=2 technical replicates, two-tailed Student t-test; ****,p<0.0001; bars represent mean±s.e.m.).

FIG. 38A is a circos plot showing location of human SNPs detected withC2c2. The assay conducted in accordance with certain example embodimentscan distinguish between human SNPs. FIG. 38B shows graphs demonstratingthat SHERLOCK can correctly genotype four different individuals at fourdifferent SNP sites in the human genome. The genotypes for eachindividual and identities of allele-sensing crRNAs are annotated beloweach plot (n=4 technical replicates; two-tailed Student t-test; *,p<0.05; **, p<0.01; ***, p<0.001; ****, p<0.0001; bars representmean±s.e.m.). FIG. 38C is a schematic of the process for detection ofcfDNA (such as cell free DNA detection of cancer mutations) inaccordance with certain example embodiments. FIG. 38D shows examplecrRNA sequences for detecting EGFR L858R and BRAF V600E. (SEQ. I.D. Nos.177 through 182). Sequences of two genomic loci assayed for cancermutations in cell-free DNA. Shown are the target genomic sequence withthe SNP highlighted in blue and the mutant/wildtype sensing crRNAsequences with synthetic mismatches colored in red.

FIGS. 39A and 39B show graphs demonstrating that C2c2 can detect themutant minor allele in mock cell-free DNA samples from the EGFR L858R(FIG. 39A) or from the BRAF V600E (FIG. 39B) minor allele. (n=4technical replicates, two tailed Student t-test; *, p<0.05; **, p<0.01,****, P<0.0001; bars represent ±s.e.m.)

FIG. 40A is a graph demonstrating that the assay can distinguish betweengenotypes at rs5082 (n=4 technical replicates; *, p<0.05; **, p<0.01;***, p<0.001; ****, p<0.0001; bars represent mean±s.e.m.). FIG. 40B is agraph demonstrating that the assay can distinguish between genotypes atrs601338 in gDNA directly from centrifuged, denatured, and boiled saliva(n=3 technical replicates; *, p<0.05; bars represent mean±s.e.m.).

FIG. 41A is a schematic of an example embodiment performed on ssDNA 1 inthe background of a target that differs from ssDNA 1 by only a singlemismatch. FIG. 41B illustrates that the assay achieves single nucleotidespecificity detection of ssDNA 1 in the presence of mismatchedbackground (target that differs by only a single mismatch from ssDNA).Various concentrations of target DNA were combined with a backgroundexcess of DNA with one mismatch and detected by the assay.

FIG. 42 is a graph showing a masking construct with a different dye Cy5also allows for effective detection.

FIG. 43 is a schematic of a gold nanoparticle colorimetric based assay.AuNPs are aggregated using a combination of DNA linkers and an RNAbridge. Upon addition of RNase activity the ssRNA bridge is cleaved andthe AuNPs are released, causing a characteristic color shift toward red.

FIG. 44 is a graph showing the ability to detect the shift in color ofdispersed nanoparticles at 520 nm. The nanoparticles were based on theexample embodiment shown in FIG. 43 and dispersed using addition ofRNase A at varying concentrations.

FIG. 45 is a set of graphs showing that the RNase colorimetric test isquantitative.

FIG. 46 is a picture of a microwell plate showing that the color shiftin the dispersed nanoparticle is visually detectable.

FIG. 47 is a set of pictures demonstrating that the colorimetric shiftis visible on a paper substrate. The test was performed for 10 minutesat 37 degrees C. on glass fiber 934-AH.

FIG. 48A is a schematic of a conformation switching aptamer inaccordance with certain example embodiments for detection of protein orsmall molecules. FIG. 48B shows the ligated product used as a completetarget for the RNA-targeting effector, which cannot detect the unligatedinput product (SEQ. I.D. Nos. 202 and 424).

FIG. 49 is an image of a gel showing that aptamer-based ligation cancreate RPA-detectable substrates. Aptamers were incubated with variouslevels of thrombin and then ligated with probe. Ligated constructs wereused as templates for a 3 minute RPA reaction. 500 nM thrombin hassignificantly higher levels of amplified target than background.

FIG. 50 shows the amino acid sequence of the HEPN domains of selectedC2c2 orthologues (SEQ. I.D. Nos. 204-233).

FIG. 51 Cas13a detection of RNA with RPA amplification (SHERLOCK) candetect ssRNA target at concentrations down to −2 aM, more sensitive thanCas13a alone (n=4 technical replicates; bars represent mean±s.e.m.).

FIGS. 52A and 52B illustrate that Cas13a detection can be used to senseviral and bacterial pathogens. FIG. 52A shows a schematic of SHERLOCKdetection of ZIKV RNA isolated from human clinical samples. FIG. 52B isa graph showing that SHERLOCK is capable of highly sensitive detectionof human ZIKV-positive serum (S) or urine (U) samples. Approximateconcentrations of ZIKV RNA shown were determined by qPCR. (n=4 technicalreplicates, two-tailed Student t-test; ****, p<0.0001; bars representmean±s.e.m.; n.d., not detected).

FIG. 53—Comparison of detection of ssRNA 1 by NASBA with primer set 2(of FIG. 11) and SHERLOCK. (n=2 technical replicates; bars representmean±s.e.m.)

FIGS. 54A-54C illustrate nucleic acid amplification with RPA andsingle-reaction SHERLOCK. FIG. 54A is a graph showing digital-dropletPCR quantitation of ssRNA 1. Adjusted concentrations for the dilutionsbased on the ddPCR results are shown above bar graphs. FIG. 54B is agraph showing PCR quantitation of ssDNA 1. Adjusted concentrations forthe dilutions based on the ddPCR results are shown above bar graphs.FIG. 54C is a graph showing that RPA, T7 transcription, and Cas13adetection reactions are compatible and achieve single molecule detectionof DNA 2 when incubated simultaneously\ (n=3 technical replicates,two-tailed Student t-test; n.s., not significant; **, p<0.01; ****,p<0.0001; bars represent mean±s.e.m.).

FIGS. 55A-55F show comparison of SHERLOCK to other sensitive nucleicacid detection tools. FIG. 55A is a graph showing detection analysis ofssDNA 1 dilution series with digital-droplet PCR (n=4 technicalreplicates, two-tailed Student t-test; n.s., not significant; *, p<0.05;**, p<0.01; ****, p<0.0001; red lines represent mean, bars representmean±s.e.m. Samples with measured copy/μL below 10-1 not shown). FIG.55B is a graph showing detection analysis of ssDNA 1 dilution serieswith quantitative PCR (n=16 technical replicates, two-tailed Studentt-test; n.s., not significant; **, p<0.01; ****, p<0.0001; red linesrepresent mean, bars represent mean±s.e.m. Samples with relative signalbelow 10-10 not shown). FIG. 55C is a graph showing detection analysisof ssDNA 1 dilution series with RPA with SYBR Green II (n=4 technicalreplicates, two-tailed Student t-test; *, p<0.05; **, p<0.01; red linesrepresent mean, bars represent mean±s.e.m. Samples with relative signalbelow 100 not shown). FIG. 55D is a graph showing detection analysis ofssDNA 1 dilution series with SHERLOCK (n=4 technical replicates,two-tailed Student t-test; **, p<0.01; ****, p<0.0001; red linesrepresent mean, bars represent mean±s.e.m. Samples with relative signalbelow 100 not shown). FIG. 55E is a graph showing percent coefficient ofvariation for a series of ssDNA 1 dilutions for four types of detectionmethods. FIG. 55F is a graph showing mean percent coefficient ofvariation for the 6e2, 6e1, 6e0, and 6e-1 ssDNA 1 dilutions for fourtypes of detection methods (bars represent mean±s.e.m.).

FIG. 56—Detection of carbapanem resistance in clinical bacterialisolates. Detection of two different carbapenem-resistance genes (KPCand NDM-1) from five clinical isolates of Klebsiella pneumoniae and anE. coli control (n=4 technical replicates, two tailed Student t-test;****, p<0.0001; bars represent mean±s.e.m.; n.d., not detected).

FIGS. 57A-57G illustrate characterization of LwCas13a sensitivity totruncated spacers and single mismatches in the target sequence. FIG. 57Ashows sequences of truncated spacer crRNAs (SEQ. I.D. Nos. 425-436) usedin FIGS. 57B to 57G. Also shown are sequences of ssRNA 1 and 2, whichhas a single base-pair difference highlighted in red. crRNAs containingsynthetic mismatches are displayed with mismatch positions colored inred. FIG. 57B is a graph showing collateral cleavage activity on ssRNA 1and 2 for 28 nt spacer crRNA with synthetic mismatches at positions 1-7(n=4 technical replicates; bars represent mean±s.e.m.). FIG. 57C is agraph showing specificity ratios of crRNA tested in FIG. 57B.Specificity ratios are calculated as the ratio of the on-target RNA(ssRNA 1) collateral cleavage to the off-target RNA (ssRNA 2) collateralcleavage. (n=4 technical replicates; bars represent mean±s.e.m.) FIG.57D is a graph showing collateral cleavage activity on ssRNA 1 and 2 for23 nt spacer crRNA with synthetic mismatches at positions 1-7 (n=4technical replicates; bars represent mean±s.e.m.). FIG. 57E is a graphshowing specificity ratios of crRNA tested in FIG. 57D. Specificityratios are calculated as the ratio of the on-target RNA (ssRNA 1)collateral cleavage to the off-target RNA (ssRNA 2) collateral cleavage(n=4 technical replicates; bars represent mean±s.e.m.). FIG. 57F is agraph showing collateral cleavage activity on ssRNA 1 and 2 for 20 ntspacer crRNA with synthetic mismatches at positions 1-7 (n=4 technicalreplicates; bars represent mean±s.e.m.). FIG. 57G is a graph showingspecificity ratios of crRNA tested in FIG. 57F. Specificity ratios arecalculated as the ratio of the on-target RNA (ssRNA 1) collateralcleavage to the off-target RNA (ssRNA 2) collateral cleavage (n=4technical replicates; bars represent mean±s.e.m.).

FIGS. 58A-58C illustrate the identification of ideal synthetic mismatchposition relative to mutations in the target sequence. FIG. 58A showssequences for evaluation of the ideal synthetic mismatch position todetect a mutation between ssRNA 1 and ssRNA (SEQ. I.D. Nos. 437-462). Oneach of the targets, crRNAs with synthetic mismatches at the colored(red) locations are tested. Each set of synthetic mismatch crRNAs isdesigned such that the mutation location is shifted in position relativeto the sequence of the spacer. Spacers are designed such that themutation is evaluated at positions 3, 4, 5, and 6 within the spacer.FIG. 58B is a graph showing collateral cleavage activity on ssRNA 1 and2 for crRNAs with synthetic mismatches at varying positions. There arefour sets of crRNAs with the mutation at either position 3, 4, 5, or 6within the spacer:target duplex region (n=4 technical replicates; barsrepresent mean±s.e.m.). FIG. 58C is a graph showing specificity ratiosof crRNA tested in FIG. 58B. Specificity ratios are calculated as theratio of the on-target RNA (ssRNA 1) collateral cleavage to theoff-target RNA (ssRNA 2) collateral cleavage (n=4 technical replicates;bars represent mean±s.e.m.).

FIG. 59—Genotyping with SHERLOCK at an additional locus and directgenotyping from boiled saliva. SHERLOCK can distinguish betweengenotypes at the rs601338 SNP site in genomic DNA directly fromcentrifuged, denatured, and boiled saliva (n=4 technical replicates,two-tailed Student t-test; **, p<0.01; ****, p<0.001; bars representmean±s.e.m.).

FIGS. 60A-60E illustrate development of synthetic genotyping standardsto accurately genotype human SNPs. FIG. 60A is a graph showinggenotyping with SHERLOCK at the rs601338 SNP site for each of the fourindividuals compared against PCR-amplified genotype standards (n=4technical replicates; bars represent mean±s.e.m.). FIG. 60B is a graphillustrating genotyping with SHERLOCK at the rs4363657 SNP site for eachof the four individuals compared against PCR-amplified genotypestandards (n=4 technical replicates; bars represent mean±s.e.m.). FIG.60C shows heatmaps of computed p-values between the SHERLOCK results foreach individual and the synthetic standards at the rs601338 SNP site. Aheatmap is shown for each of the allele-sensing crRNAs. The heatmapcolor map is scaled such that insignificance (p>0.05) is red andsignificance (p<0.05) is blue (n=4 technical replicates, one-way ANOVA).FIG. 60D shows heatmaps of computed p-values between the SHERLOCKresults for each individual and the synthetic standards at the rs4363657SNP site. A heatmap is shown for each of the allele-sensing crRNAs. Theheatmap color map is scaled such that insignificance (p>0.05) is red andsignificance (p<0.05) is blue (n=4 technical replicates, one-way ANOVA).FIG. 60E is a guide for understanding the p-value heatmap results ofSHERLOCK genotyping. Genotyping can easily be called by choosing theallele that corresponds to a p-value>0.05 between the individual andallelic synthetic standards. Red blocks correspond to non-significantdifferences between the synthetic standard and individual's SHERLOCKresult and thus a genotype-positive result. Blue blocks correspond tosignificant differences between the synthetic standard and individual'sSHERLOCK result and thus a genotype-negative result.

FIG. 61—Detection of ssDNA 1 as a small fraction of mismatchedbackground target. SHERLOCK detection of a dilution series of ssDNA 1 ona background of human genomic DNA. Note that there should be no sequencesimilarity between the ssDNA 1 target being detected and the backgroundgenomic DNA (n=2 technical replicates; bars represent mean±s.e.m.).

FIG. 62A is a graph showing urine samples from patients with Zika virusthat were heat inactivated for 5 minutes at 95° C. FIG. 62B is a graphshowing serum samples from patients with Zika virus that were heatinactivated for 5 minutes at 65° C. One microliter of inactivated urineor serum was used as input for a 2 hr RPA reaction followed by a 3 hourC2c2/Cas13a detection reaction, in accordance with an exampleembodiment. Error bars indicate 1 SD based on n=4 technical replicatesfor the detection reaction.

FIGS. 63A, 63B. Urine samples from patients with Zika virus wereheat-inactivated for 5 minutes at 95° C. One microliter of inactivatedurine was used as input for a 30 minute RPA reaction followed by a 3hour (FIG. 63A) or 1 hour (FIG. 63B) C2c2/Cas13 detection reaction, inaccordance with example embodiments. Error bars indicate 1 SD based onn=4 technical replicates for the detection reaction.

FIG. 64—Urine samples from patients with Zika virus wereheat-inactivated for 5 minutes at 95° C. One microliter of inactivatedurine was used as input for a 20 minute RPA reaction followed by a 1hour C2c2/Cas13a detection reaction. Healthy human urine was used as anegative control. Error bars indicate 1 SD based on n=4 technicalreplicates or the detection reaction.

FIGS. 65A, 65B—Urine samples from patients with Zika virus wereheat-inactivated for 5 minutes at 95° C. One microliter of inactivatedurine was used as input for a 20 minute RPA reaction followed by a 1hour C2c2/Cas13a detection reaction in the presence or absence of guideRNA. Data are normalized by subtracting the average fluorescence valuesfor no-guide detection reactions from the detection reactions containingguides. Healthy human urine was used as a negative control. Error barsindicate 1 SD based on n=4 technical replicates for the detectionreaction.

FIG. 66—Shows detection of two malaria specific targets with fourdifferent guide RNA designs, in accordance with example embodiments(SEQ. I.D. Nos. 463-474).

FIGS. 67A, 67B—graphs showing editing preferences of different Cas13borthologs using (FIG. 67A) Poly U, Poly C, Rnase Alert, (FIG. 67B) PolyU, Poly A, and Poly G fluorescent sensors. See Table 3 for key.

FIG. 68—The panel on the left provides a schematic of a multiplex assayusing different Cas13b orthologs with different editing preferences, andthe panel on the right provides data demonstrating the feasibility ofsuch an assay using Cas13b10 and Cas13b5.

FIG. 69—provides graphs showing dual multiplexing with Cas13b5(Prevotella sp. MA2106) and Cas13b9 (Prevotella intermedia) orthologues.Both effector proteins and guide sequences were contained in the samereaction allowing for dual multiplexing in the same reaction usingdifferent fluorescent readouts (poly U 530 nm and poly A 485 nm).

FIG. 70—provides same as FIG. 69 but in this instance using Cas13a(Leptorichia wadei LwaCas13a) orthologs and Cas13b orthologs (Prevotellasp. MA2016, Cas13b5).

FIG. 71—provides a method for tiling target sequences with multipleguide sequences in order to determine robustness of targeting, inaccordance with certain example embodiments (SEQ. I.D. Nos. 475 and476).

FIG. 72—provides hybrid chain reaction (HCR) gels showing that Cas13effector proteins may be used to unlock an initiator, for an example aninitiator incorporated in a masking construct as described herein, toactivate a hybridization chain reaction.

FIG. 73—provides data showing the ability to detect Pseudomonasaeruginosa in complex lysate.

FIG. 74—provides data showing ion preferences of certain Cas13orthologues in accordance with certain example embodiments. All targetconcentrations were 20 nM input with ion concentrations of (1 mM and 10mM).

FIG. 75—provides data showing that Cas13b12 has a 1 mM Zinc sulfatepreference for cleavage.

FIG. 76—provides data showing buffer optimization may boost signal tonoise of Cas13b5 on a polyA reporter. Old buffer comprises 40 mMTris-HCL, 60 mM NaCl, 6 mM MgCl2, pH 7.3. New buffer comprises 20 mMHEPES pH 6.8, 6 mM MgCl2 and 60 mM NaCl.

FIG. 77—provides a schematic of type VI-A/C Crispr systems and TypeVI-B1 and B2 systems as well as a phylogenetic tree of representativeCas13b orthologues.

FIG. 78—provides relative cleavage activity at different nucleotides ofvarious Cas13b orthologs and relative to a LwCas13a.

FIG. 79—provides a graph showing relative sensitivity of various exampleCas13 orthologs.

FIG. 80—provides a graph showing the ability to achieve zepto molar (zM)levels of detection using an example embodiment.

FIG. 81A—provides schematic of a multiplex assay using Cas13 orthologswith different editing preferences and polyN based masking constructs.FIG. 81B shows a graph of multiplex detection of Dengue and Zika targetsusing Cas13b5 and Cas13b10 respectively. FIG. 81C provides a heatmapshowing multiplex detection of Zika ssRNA and Dengue ssRNA targets atvarying concentrations. FIG. 81D provides a graph showing multiplexdetection of specific genotypes at varying crRNA ratios.

FIGS. 82A-82F provide data showing results of primer optimizationexperiments and detection of pseudomonas over a range of conditions.

FIGS. 83A-83H illustrate the biochemical characterization of the Cas13bfamily of RNA-guided RNA-targeting enzymes and increased sensitivity andquantitative SHERLOCK. FIG. 83A shows a schematic of the CRISPR-Cas13loci and crRNA structure. FIG. 83B is a heatmap of the base preferenceof 15 Cas13b orthologs targeting ssRNA 1 with sensor probes consistingof a hexamer homopolymer of A, C, G, or U bases. FIG. 83C is a schematicof cleavage motif preference discovery screen and preferred two-basemotifs for LwaCas13a and PsmCas13b. Values represented in the heatmapare the counts of each two-base across all depleted motifs. Motifs areconsidered depleted if the −log ₂(target/no target) value is above 1.0in the LwaCas13a condition or 0.5 in the PsmCas13b condition. In the−log ₂(target/no target) value, target and no target denote thefrequency of a motif in the target and no target conditions,respectively. FIG. 83D is a graph showing orthogonal base preferences ofPsmCas13b and LwaCas13a targeting ssRNA 1 with either a U6 or A6 sensorprobe. FIG. 83E illustrates graphs showing single molecule SHERLOCKdetection with LwaCas13a and PsmCas13b targeting Dengue ssRNA target.FIG. 83F illustrates graphs showing single molecule SHERLOCK detectionwith LwaCas13a and PsmCas13b in large reaction volumes for increasedsensitivity targeting ssRNA target 1. FIG. 83G is a graph showingquantitation of P. aeruginosa synthetic DNA at various RPA primerconcentrations. FIG. 83H is a graph showing correlation of P. aeruginosasynthetic DNA concentration with detected fluorescence.

FIGS. 84A-84H illustrate in-sample multiplexing SHERLOCK with orthogonalCas13 enzymes. FIG. 84A is a schematic of in-sample multiplexing usingorthogonal Cas13 enzymes. FIG. 84B is a graph showing in-samplemultiplexed detection of 20 nM Zika and Dengue synthetic RNA withLwaCas13a and PsmCas13b collateral activity. FIG. 84C illustratesheatmaps showing multiplexed RPA and collateral detection at decreasingconcentrations of S. aureus thermonuclease and P. aeruginosaacyltransferase synthetic targets with LwaCas13a and PsmCas13b. FIG. 84Dis a graph showing multiplexed genotyping with human samples at rs601338with LwaCas13a and CcaCas13b. FIG. 84E is a schematic of theranostictimeline for detection of disease alleles, correction with REPAIR, andassessment of REPAIR correction. FIG. 84F is a graph showing in-samplemultiplexed detection of APC alleles from healthy- anddisease-simulating samples with LwaCas13a and PsmCas13b. FIG. 84G is agraph showing quantitation of REPAIR editing efficiency at the targetedAPC mutation. FIG. 84H is a graph showing multiplexed detection of APCalleles from REPAIR targeting and non-targeting samples with LwaCas13aand PsmCas13b.

FIG. 85—provides a tree of 15 Cas13b orthologs purified and evaluatedfor in vitro collateral activity. Cas13b gene (blue), Csx27/Csx28 gene(red/yellow), and CRISPR array (grey) are shown.

FIGS. 86A-86C illustrate protein purification of Cas13 orthologs. FIG.86A shows chromatograms of size exclusion chromatography for Cas13b,LwCas13a and LbaCas13a used in this study. Measured UV absorbance (mAU)is shown against the elution volume (ml). FIG. 86B shows an SDS-PAGE gelof purified Cas13b orthologs. Fourteen Cas13b orthologs are loaded fromleft to right. A protein ladder is shown to the left. FIG. 86C shows afinal SDS-PAGE gel of LbaCas13a dilutions (right) and BSA standardtitration (left). Five dilutions of BSA and two of LbaCas13 are shown.

FIGS. 87A-87D show graphs illustrating base preference of Cas13bortholog collateral cleavage. FIG. 87A is a graph showing cleavageactivity of fourteen Cas13b orthologs targeting ssRNA 1 using ahomopolymer adenine sensor six nucleotides long. FIG. 87B is a graphshowing cleavage activity of fourteen Cas13b orthologs targeting ssRNA 1using a homopolymer uridine sensor six nucleotides long. FIG. 87C is agraph showing cleavage activity of fourteen Cas13b orthologs targetingssRNA 1 using a homopolymer guanine sensor six nucleotides long. FIG.87D is a graph showing cleavage activity of fourteen Cas13b orthologstargeting ssRNA 1 using a homopolymer cytidine sensor six nucleotideslong.

FIG. 88—shows size analysis of random motif-library after Cas13collateral cleavage. Bioanalyzer traces for LwaCas13a-, PsmCas13b-,CcaCas13b-, and RNase A-treated library samples showing changes inlibrary size after RNase activity. Cas13 orthologs are targeting DenguessRNA and cleave the random motif-library due to collateral cleavage.Marker standards are shown in the first lane.

FIGS. 89A-89D show a representation of various motifs after cleavage byRNases. FIG. 89A shows box plots showing motif distribution of target tono-target ratios for LwaCas13a, PsmCas13b, CcaCas13b, and RNase A at 5minute and 60 minute timepoints. RNase A ratios were compared to theaverage of the three Cas13 no-target conditions. Ratios are also anaverage of two cleavage reaction replicates. FIG. 89B is a graph showingnumber of enriched motifs for LwaCas13a, PsmCas13b, CcaCas13b, and RNaseA at the 60 minute timepoint. Enrichment motif was calculated as motifsabove −log ₂(target/no target) thresholds of either 1 (LwaCas13a,CcaCas13b, and RNase A) or 0.5 (PsmCas13b). A threshold of 1 correspondsto at least 50% depletion while a threshold of 0.5 corresponds to atleast 30% depletion. FIG. 89C shows sequence logos generated fromenriched motifs for LwaCas13a, PsmCas13b, and CcaCas13b. LwaCas13a andCcaCas13b show a strong U preference as would be expected, whilePsmCas13b shows a unique preference for A bases across the motif, whichis consistent with homopolymer collateral activity preferences. FIG. 89Dis a heatmap showing the orthogonal motif preferences of LwaCas13a,PsmCas13b, and CcaCas13b. Values represented in the heatmap are the −log₂(target/no target) value of each shown motif. In the −log₂(target/notarget) value, target and no target denote the frequency of a motif inthe target and no target conditions, respectively.

FIGS. 90A-90C show single-base and two-base preferences of RNasesdetermined by random motif library screen. FIG. 90A illustrates heatmapsshowing single base preferences for LwaCas13a, PsmCas13b, CcaCas13b, andRNase A at the 60 minute timepoint as determined by the random motiflibrary cleavage screen. Values represented in the heatmap are thecounts of each base across all depleted motifs. Motifs are considereddepleted if the −log₂(target/no target) value is above 1.0 in theLwaCas13a, CcaCas13b, and RNase A conditions or 0.5 in the PsmCas13bcondition. In the −log ₂(target/no target) value, target and no targetdenote the frequency of a motif in the target and no target conditions,respectively.

FIG. 90B is a heatmap showing two-base preference for CcaCas13b asdetermined by the random motif library cleavage screen. Valuesrepresented in the heatmap are the counts of each 2-base across alldepleted motifs. Motifs are considered depleted if the −log ₂(target/notarget) value is above 1.0 in the LwaCas13a, CcaCas13b, and RNase Aconditions or 0.5 in the PsmCas13b condition. In the −log ₂(target/notarget) value, target and no target denote the frequency of a motif inthe target and no target conditions, respectively. FIG. 90C is a heatmapshowing two-base preference for RNase A as determined by the randommotif library cleavage screen. Values represented in the heatmap are thecounts of each two-base across all depleted motifs. Motifs areconsidered depleted if the −log ₂(target/no target) value is above 1.0in the LwaCas13a, CcaCas13b, and RNase A conditions or 0.5 in thePsmCas13b condition. In the −log₂(target/no target) value, target and notarget denote the frequency of a motif in the target and no targetconditions, respectively.

FIG. 91—illustrates three-base preferences of RNases determined byrandom motif library screen. Heatmaps show three-base preferences forLwaCas13a, PsmCas13b, CcaCas13b, and RNase A at the 60 minute timepointas determined by the random motif library cleavage screen. Valuesrepresented in the heatmap are the counts of each 3-base across alldepleted motifs. Motifs are considered depleted if the −log ₂(target/notarget) value is above 1.0 in the LwaCas13a, CcaCas13b, and RNase Aconditions or 0.5 in the PsmCas13b condition. In the −log ₂(target/notarget) value, target and no target denote the frequency of a motif inthe target and no target conditions, respectively.

FIGS. 92A-92D illustrate four-base preferences of RNases determined byrandom motif library screen. Heatmaps show four-base preferences for(FIG. 92B) LwaCas13a, (FIG. 92C) PsmCas13b, (FIG. 92A) CcaCas13b, and(FIG. 92D) RNase A at the 60 minute timepoint as determined by therandom motif library cleavage screen. Values represented in the heatmapare the counts of each 4-base across all depleted motifs. Motifs areconsidered depleted if the −log ₂(target/no target) value is above 1.0in the LwaCas13a, CcaCas13b, and RNase A conditions or 0.5 in thePsmCas13b condition. In the −log ₂(target/no target) value, target andno target denote the frequency of a motif in the target and no targetconditions, respectively.

FIGS. 93A-93C show results of testing base cleavage preferences of Cas13orthologs with in vitro cleavage of poly-X substrates. FIG. 93Aillustrates in vitro cleavage of poly-U, C, G, and A targets withLwaCas13a incubated with and without crRNA. FIG. 93B illustrates invitro cleavage of poly-U, C, G, and A targets with CcaCas13b incubatedwith and without crRNA. FIG. 93C illustrates in vitro cleavage ofpoly-U, C, G, and A targets with PsmCas13b incubated with and withoutcrRNA.

FIGS. 94A and 94B show graphs of results of buffer optimization ofPsmCas13b cleavage activity. FIG. 94A. A variety of buffers are testedfor their effect on PsmCas13b collateral activity after targetingssRNA 1. FIG. 94B. The optimized buffer is compared to the originalbuffer at different PsmCas13b-crRNA complex concentrations.

FIGS. 95A-95F are graphs illustrating ion preference of Cas13 orthologsfor collateral cleavage. FIG. 95A. Cleavage activity of PsmCas13b with afluorescent poly U sensor for divalent cations Ca, Co, Cu, Mg, Mn, Ni,and Zn. PsmCas13b is incubated with a crRNA targeting a synthetic DenguessRNA. FIG. 95B. Cleavage activity of PsmCas13b with a fluorescent polyA sensor for divalent cations Ca, Co, Cu, Mg, Mn, Ni, and Zn. PsmCas13bis incubated with a crRNA targeting a synthetic Dengue ssRNA. FIG. 95C.Cleavage activity of Pin2Cas13b with a fluorescent poly U sensor fordivalent cations Ca, Co, Cu, Mg, Mn, Ni, and Zn. Pin2Cas13b is incubatedwith a crRNA targeting a synthetic Dengue ssRNA. FIG. 95D. Cleavageactivity of Pin2Cas13b with a fluorescent poly A sensor for divalentcations Ca, Co, Cu, Mg, Mn, Ni, and Zn. Pin2Cas13b is incubated with acrRNA targeting a synthetic Dengue ssRNA. FIG. 95E. Cleavage activity ofCcaCas13b with a fluorescent poly U sensor for divalent cations Ca, Co,Cu, Mg, Mn, Ni, and Zn. CcaCas13b is incubated with a crRNA targeting asynthetic Dengue ssRNA. FIG. 95F. Cleavage activity of CcaCas13b with afluorescent poly A sensor for divalent cations Ca, Co, Cu, Mg, Mn, Ni,and Zn. CcaCas13b is incubated with a crRNA targeting a synthetic DenguessRNA.

FIGS. 96A and 96B are graphs showing comparison of cleavage activity forCas13 orthologs with adenine cleavage preference. FIG. 96A. Cleavageactivity of PsmCas13b and LbaCas13a incubated with respective crRNAstargeting a synthetic Zika target at different concentrations (n=4technical replicates, two-tailed Student t-test; n.s., not significant;*, p<0.05; **, p<0.01; ***, p<0.001; ****, p<0.0001; bars representmean±s.e.m.). FIG. 96B. Cleavage activity of PsmCas13b and LbaCas13aincubated with respective crRNAs targeting a synthetic Dengue target atdifferent concentrations (n=4 technical replicates, two-tailed Studentt-test; n.s., not significant; *, p<0.05; **, p<0.01; ***, p<0.001;****, p<0.0001; bars represent mean±s.e.m.).

FIGS. 97A and 97B are graphs illustrating attomolar detection of ZikassRNA target 4 with SHERLOCK with LwaCas13a and PsmCas13b. FIG. 97A.SHERLOCK detection of Zika ssRNA at different concentrations withLwaCas13a and poly U sensor. FIG. 97B. SHERLOCK detection of Zika ssRNAat different concentrations with PsmCas13b and poly A sensor.

FIG. 98—illustrates attomolar detection of Dengue ssRNA with SHERLOCK atdifferent concentrations of CcaCas13b.

FIGS. 99A and 99B are graphs showing results from testing Cas13 orthologreprogrammability with crRNAs tiling ssRNA 1. FIG. 99A. Cleavageactivity of LwaCas13a and CcaCas13b with crRNAs tiled across ssRNA1.FIG. 99B. Cleavage activity of PsmCas13b with crRNAs tiled acrossssRNA1.

FIGS. 100A and 100B are graphs showing the effect of crRNA spacer lengthon Cas13 ortholog cleavage. FIG. 100A. Cleavage activity of PsmCas13bwith ssRNA1-targeting crRNAs of varying spacer lengths. FIG. 100B.Cleavage activity of CcaCas13b with ssRNA1-targeting crRNAs of varyingspacer lengths.

FIGS. 101A-101G illustrate optimizing primer concentration forquantitative SHERLOCK. FIG. 101A. SHERLOCK kinetic curves of LwaCas13aincubated with Zika RNA targets of different concentration and acomplementary crRNA at an RPA primer concentration of 480 nM. FIG. 101B.SHERLOCK kinetic curves of LwaCas13a incubated with Zika RNA targets ofdifferent concentration and a complementary crRNA at an RPA primerconcentration of 240 nM. FIG. 101C. SHERLOCK kinetic curves of LwaCas13aincubated with Zika RNA targets of different concentration and acomplementary crRNA at an RPA primer concentration of 120 nM. FIG. 101D.SHERLOCK kinetic curves of LwaCas13a incubated with Zika RNA targets ofdifferent concentration and a complementary crRNA at an RPA primerconcentration of 24 nM. FIG. 101E. SHERLOCK detection of Zika RNA ofdifferent concentrations with four different RPA primer concentrations:480 nM, 240 nM, 120 nM, 60 nM, and 24 nM. FIG. 101F. The mean R²correlation between background subtracted fluorescence of SHERLOCK andthe Zika target RNA concentration at different RPA primerconcentrations. FIG. 101G. Quantitative SHERLOCK detection of Zika RNAtargets at different concentrations in a 10-fold dilution series (blackpoints) and 2-fold dilution series (red points). An RPA primerconcentration of 120 nM was used.

FIGS. 102A-102C illustrate multiplexed detection of Zika and Denguetargets. FIG. 102A. Multiplexed two-color detection using LwaCas13atargeting a Zika ssRNA target and PsmCas13b targeting a Dengue ssRNAtarget. Both targets are at 20 nM input. All Data shown represent 180minutes time point of reaction. FIG. 102B. Multiplexed two-colordetection using LwaCas13a targeting a Zika ssRNA target and PsmCas13btargeting a Dengue ssRNA target. Both targets are at 200 pM input. FIG.102C. In-sample multiplexed detection of 20 pM Zika and Dengue syntheticRNA with CcaCas13a and PsmCas13b collateral activity.

FIGS. 103A and 103B are heatmaps illustrating in-sample multiplexed RNAdetection of (FIG. 103B) Zika and (FIG. 103A) Dengue ssRNA. In-samplemultiplexed RPA and collateral detection at decreasing concentrations ofZika and Dengue synthetic targets with PsmCas13b and CcaCas13b.

FIGS. 104A and 104B are graphs illustrating non-multiplexed theranosticdetection of mutations and REPAIR editing. FIG. 104A. Detection of APCalleles from healthy- and disease-simulated samples with LwaCas13a. FIG.104B. Detection with LwaCas13a of editing correction at the APC allelesfrom REPAIR targeting and non-targeting samples.

FIGS. 105A-105E illustrate colorimetric detection of RNase activity withgold nanoparticle aggregation. FIG. 105A is a schematic ofgold-nanoparticle based colorimetric readout for RNase activity. In theabsence of RNase activity, RNA linkers aggregate gold nanoparticles,leading to loss of red color. Cleavage of RNA linkers releasesnanoparticles and results in a red color change. FIG. 105B is an imageof colorimetric reporters after 120 minutes of RNase digestion atvarious units of RNase A. FIG. 105C is a graph showing kinetics at 520nm absorbance of AuNP colorimetric reporters with digestion at variousunit concentrations of RNase A. FIG. 105D is a graph showing 520 nmabsorbance of AuNP colorimetric reporters after 120 minutes of digestionat various unit concentrations of RNase A. FIG. 105E is a graph showingtime to half-A520 maximum of AuNP colorimetric reporters with digestionat various unit concentrations of RNase A.

FIGS. 106A-106C illustrate quantitative detection of CP4-EPSPS gene fromsoybean genomic DNA. FIG. 106A is a graph showing mean correlation R² ofthe SHERLOCK background subtracted fluorescence and CP4-EPSPS beanpercentage at different time points of detection. Bean percentagedepicts the amount of round-up ready beans in a mixture of round-upready and wild-type beans. The CP4-EPSPS gene is only present inround-up ready beans. FIG. 106B is a graph showing SHERLOCK detection ofCP4-EPSPS resistance gene at different bean percentages showing thequantitative nature of SHERLOCK detection at 30 minutes of incubation.FIG. 106C is a graph showing SHERLOCK detection of Lectin gene atdifferent bean percentages. Bean percentage depicts the amount ofround-up ready beans in a mixture of round-up ready and wild-type beans.The Lectin gene is present in both types of beans and therefore shows nocorrelation to round-up ready bean percentage.

DETAILED DESCRIPTION OF THE EXAMPLE EMBODIMENTS General Definitions

Unless defined otherwise, technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this disclosure pertains. Definitions of common termsand techniques in molecular biology may be found in Molecular Cloning: ALaboratory Manual, 2nd edition (1989) (Sambrook, Fritsch, and Maniatis);Molecular Cloning: A Laboratory Manual, 4th edition (2012) (Green andSambrook); Current Protocols in Molecular Biology (1987) (F. M. Ausubelet al. eds.); the series Methods in Enzymology (Academic Press, Inc.):PCR 2: A Practical Approach (1995) (M. J. MacPherson, B. D. Hames, andG. R. Taylor eds.): Antibodies, A Laboratory Manual (1988) (Harlow andLane, eds.): Antibodies A Laboratory Manual, 2nd edition 2013 (E. A.Greenfield ed.); Animal Cell Culture (1987) (R. I. Freshney, ed.);Benjamin Lewin, Genes IX, published by Jones and Bartlet, 2008 (ISBN0763752223); Kendrew et al. (eds.), The Encyclopedia of MolecularBiology, published by Blackwell Science Ltd., 1994 (ISBN 0632021829);Robert A. Meyers (ed.), Molecular Biology and Biotechnology: aComprehensive Desk Reference, published by VCH Publishers, Inc., 1995(ISBN 9780471185710); Singleton et al., Dictionary of Microbiology andMolecular Biology 2nd ed., J. Wiley & Sons (New York, N.Y. 1994), March,Advanced Organic Chemistry Reactions, Mechanisms and Structure 4th ed.,John Wiley & Sons (New York, N.Y. 1992); and Marten H. Hofker and Janvan Deursen, Transgenic Mouse Methods and Protocols, 2nd edition (2011).

As used herein, the singular forms “a”, “an”, and “the” include bothsingular and plural referents unless the context clearly dictatesotherwise.

The term “optional” or “optionally” means that the subsequent describedevent, circumstance or substituent may or may not occur, and that thedescription includes instances where the event or circumstance occursand instances where it does not.

The recitation of numerical ranges by endpoints includes all numbers andfractions subsumed within the respective ranges, as well as the recitedendpoints.

The terms “about” or “approximately” as used herein when referring to ameasurable value such as a parameter, an amount, a temporal duration,and the like, are meant to encompass variations of and from thespecified value, such as variations of +/−10% or less, +/−5% or less,+/−1% or less, and +1-0.1% or less of and from the specified value,insofar such variations are appropriate to perform in the disclosedinvention. It is to be understood that the value to which the modifier“about” or “approximately” refers is itself also specifically, andpreferably, disclosed.

Reference throughout this specification to “one embodiment”, “anembodiment,” “an example embodiment,” means that a particular feature,structure or characteristic described in connection with the embodimentis included in at least one embodiment of the present invention. Thus,appearances of the phrases “in one embodiment,” “in an embodiment,” or“an example embodiment” in various places throughout this specificationare not necessarily all referring to the same embodiment, but may.Furthermore, the particular features, structures or characteristics maybe combined in any suitable manner, as would be apparent to a personskilled in the art from this disclosure, in one or more embodiments.Furthermore, while some embodiments described herein include some butnot other features included in other embodiments, combinations offeatures of different embodiments are meant to be within the scope ofthe invention. For example, in the appended claims, any of the claimedembodiments can be used in any combination.

“C2c2” is now referred to as “Cas13a”, and the terms are usedinterchangeably herein unless indicated otherwise.

All publications, published patent documents, and patent applicationscited herein are hereby incorporated by reference to the same extent asthough each individual publication, published patent document, or patentapplication was specifically and individually indicated as beingincorporated by reference.

Overview

Microbial Clustered Regularly Interspaced Short Palindromic Repeats(CRISPR) and CRISPR-associated (CRISPR-Cas) adaptive immune systemscontain programmable endonucleases, such as Cas9 and Cpf1 (Shmakov etal., 2017; Zetsche et al., 2015). Although both Cas9 and Cpf1 targetDNA, single effector RNA-guided RNases have been recently discovered(Shmakov et al., 2015) and characterized (Abudayyeh et al., 2016;Smargon et al., 2017), including C2c2, providing a platform for specificRNA sensing. RNA-guided RNases can be easily and convenientlyreprogrammed using CRISPR RNA (crRNAs) to cleave target RNAs. Unlike theDNA endonucleases Cas9 and Cpf1, which cleave only its DNA target,RNA-guided RNases, like C2c2, remains active after cleaving its RNAtarget, leading to “collateral” cleavage of non-targeted RNAs inproximity (Abudayyeh et al., 2016). This crRNA-programmed collateral RNAcleavage activity presents the opportunity to use RNA-guided RNases todetect the presence of a specific RNA by triggering in vivo programmedcell death or in vitro nonspecific RNA degradation that can serve as areadout (Abudayyeh et al., 2016; East-Seletsky et al., 2016).

The embodiments disclosed herein utilized RNA targeting effectors toprovide a robust CRISPR-based diagnostic with attomolar sensitivity.Embodiments disclosed herein can detect broth DNA and RNA withcomparable levels of sensitivity and can differentiate targets fromnon-targets based on single base pair differences. Moreover, theembodiments disclosed herein can be prepared in freeze-dried format forconvenient distribution and point-of-care (POC) applications. Suchembodiments are useful in multiple scenarios in human health including,for example, viral detection, bacterial strain typing, sensitivegenotyping, and detection of disease-associated cell free DNA. For easeof reference, the embodiments disclosed herein may also be referred toas SHERLOCK (Specific High-sensitivity Enzymatic Reporter unLOCKing).

In one aspect, the embodiments disclosed herein are directed to anucleic acid detection system comprising a CRISPR system, one or moreguide RNAs designed to bind to corresponding target molecules, a maskingconstruct, and optional amplification reagents to amplify target nucleicacid molecules in a sample. In certain example embodiments, the systemmay further comprise one or more detection aptamers. The one or moredetection aptamers may comprise a RNA polymerase site or primer bindingsite. The one or more detection aptamers specifically bind one or moretarget polypeptides and are configured such that the RNA polymerase siteor primer binding site is exposed only upon binding of the detectionaptamer to a target peptide. Exposure of the RNA polymerase sitefacilitates generation of a trigger RNA oligonucleotide using theaptamer sequence as a template. Accordingly, in such embodiments the oneor more guide RNAs are configured to bind to a trigger RNA.

In another aspect, the embodiments disclosed herein are directed to adiagnostic device comprising a plurality of individual discrete volumes.Each individual discrete volume comprises a CRISPR effector protein, oneor more guide RNAs designed to bind to a corresponding target molecule,and a masking construct. In certain example embodiments, RNAamplification reagents may be pre-loaded into the individual discretevolumes or be added to the individual discrete volumes concurrently withor subsequent to addition of a sample to each individual discretevolume. The device may be a microfluidic based device, a wearabledevice, or device comprising a flexible material substrate on which theindividual discrete volumes are defined.

In another aspect, the embodiments disclosed herein are directed to amethod for detecting target nucleic acids in a sample comprisingdistributing a sample or set of samples into a set of individualdiscrete volumes, each individual discrete volume comprising a CRISPReffector protein, one or more guide RNAs designed to bind to one targetoligonucleotides, and a masking construct. The set of samples are thenmaintained under conditions sufficient to allow binding of the one ormore guide RNAs to one or more target molecules. Binding of the one ormore guide RNAs to a target nucleic acid in turn activates the CRISPReffector protein. Once activated, the CRISPR effector protein thendeactivates the masking construct, for example, by cleaving the maskingconstruct such that a detectable positive signal is unmasked, released,or generated. Detection of the positive detectable signal in anindividual discrete volume indicates the presence of the targetmolecules.

In yet another aspect, the embodiments disclosed herein are directed toa method for detecting polypeptides. The method for detectingpolypeptides is similar to the method for detecting target nucleic acidsdescribed above. However, a peptide detection aptamer is also included.The peptide detection aptamers function as described above andfacilitate generation of a trigger oligonucleotide upon binding to atarget polypeptide. The guide RNAs are designed to recognize the triggeroligonucleotides thereby activating the CRISPR effector protein.Deactivation of the masking construct by the activated CRISPR effectorprotein leads to unmasking, release, or generation of a detectablepositive signal.

Crispr Effector Proteins

In general, a CRISPR-Cas or CRISPR system as used in herein and indocuments, such as WO 2014/093622 (PCT/US2013/074667), referscollectively to transcripts and other elements involved in theexpression of or directing the activity of CRISPR-associated (“Cas”)genes, including sequences encoding a Cas gene, a tracr(trans-activating CRISPR) sequence (e.g. tracrRNA or an active partialtracrRNA), a tracr-mate sequence (encompassing a “direct repeat” and atracrRNA-processed partial direct repeat in the context of an endogenousCRISPR system), a guide sequence (also referred to as a “spacer” in thecontext of an endogenous CRISPR system), or “RNA(s)” as that term isherein used (e.g., RNA(s) to guide Cas, such as Cas9, e.g. CRISPR RNAand transactivating (tracr) RNA or a single guide RNA (sgRNA) (chimericRNA)) or other sequences and transcripts from a CRISPR locus. Ingeneral, a CRISPR system is characterized by elements that promote theformation of a CRISPR complex at the site of a target sequence (alsoreferred to as a protospacer in the context of an endogenous CRISPRsystem). When the CRISPR protein is a C2c2 protein, a tracrRNA is notrequired. C2c2 has been described in Abudayyeh et al. (2016) “C2c2 is asingle-component programmable RNA-guided RNA-targeting CRISPR effector”;Science; DOI: 10.1126/science.aaf5573; and Shmakov et al. (2015)“Discovery and Functional Characterization of Diverse Class 2 CRISPR-CasSystems”, Molecular Cell, DOI: dx.doi.org/10.1016/j.molcel.2015.10.008;which are incorporated herein in their entirety by reference. Cas13b hasbeen described in Smargon et al. (2017) “Cas13b Is a Type VI-BCRISPR-Associated RNA-Guided RNases Differentially Regulated byAccessory Proteins Csx27 and Csx28,” Molecular Cell. 65, 1-13;dx.doi.org/10.1016/j.molcel.2016.12.023., which is incorporated hereinin its entirety by reference.

In certain embodiments, a protospacer adjacent motif (PAM) or PAM-likemotif directs binding of the effector protein complex as disclosedherein to the target locus of interest. In some embodiments, the PAM maybe a 5′ PAM (i.e., located upstream of the 5′ end of the protospacer).In other embodiments, the PAM may be a 3′ PAM (i.e., located downstreamof the 5′ end of the protospacer). The term “PAM” may be usedinterchangeably with the term “PFS” or “protospacer flanking site” or“protospacer flanking sequence”.

In a preferred embodiment, the CRISPR effector protein may recognize a3′ PAM. In certain embodiments, the CRISPR effector protein mayrecognize a 3′ PAM which is 5′H, wherein H is A, C or U. In certainembodiments, the effector protein may be Leptotrichia shahii C2c2p, morepreferably Leptotrichia shahii DSM 19757 C2c2, and the 3′ PAM is a 5′ H.

In the context of formation of a CRISPR complex, “target sequence”refers to a sequence to which a guide sequence is designed to havecomplementarity, where hybridization between a target sequence and aguide sequence promotes the formation of a CRISPR complex. A targetsequence may comprise RNA polynucleotides. The term “target RNA” refersto a RNA polynucleotide being or comprising the target sequence. Inother words, the target RNA may be a RNA polynucleotide or a part of aRNA polynucleotide to which a part of the gRNA, i.e. the guide sequence,is designed to have complementarity and to which the effector functionmediated by the complex comprising CRISPR effector protein and a gRNA isto be directed. In some embodiments, a target sequence is located in thenucleus or cytoplasm of a cell.

The nucleic acid molecule encoding a CRISPR effector protein, inparticular C2c2, is advantageously codon optimized CRISPR effectorprotein. An example of a codon optimized sequence, is in this instance asequence optimized for expression in eukaryotes, e.g., humans (i.e.being optimized for expression in humans), or for another eukaryote,animal or mammal as herein discussed; see, e.g., SaCas9 human codonoptimized sequence in WO 2014/093622 (PCT/US2013/074667). Whilst this ispreferred, it will be appreciated that other examples are possible andcodon optimization for a host species other than human, or for codonoptimization for specific organs is known. In some embodiments, anenzyme coding sequence encoding a CRISPR effector protein is a codonoptimized for expression in particular cells, such as eukaryotic cells.The eukaryotic cells may be those of or derived from a particularorganism, such as a plant or a mammal, including but not limited tohuman, or non-human eukaryote or animal or mammal as herein discussed,e.g., mouse, rat, rabbit, dog, livestock, or non-human mammal orprimate. In some embodiments, processes for modifying the germ linegenetic identity of human beings and/or processes for modifying thegenetic identity of animals which are likely to cause them sufferingwithout any substantial medical benefit to man or animal, and alsoanimals resulting from such processes, may be excluded. In general,codon optimization refers to a process of modifying a nucleic acidsequence for enhanced expression in the host cells of interest byreplacing at least one codon (e.g. about or more than about 1, 2, 3, 4,5, 10, 15, 20, 25, 50, or more codons) of the native sequence withcodons that are more frequently or most frequently used in the genes ofthat host cell while maintaining the native amino acid sequence. Variousspecies exhibit particular bias for certain codons of a particular aminoacid. Codon bias (differences in codon usage between organisms) oftencorrelates with the efficiency of translation of messenger RNA (mRNA),which is in turn believed to be dependent on, among other things, theproperties of the codons being translated and the availability ofparticular transfer RNA (tRNA) molecules. The predominance of selectedtRNAs in a cell is generally a reflection of the codons used mostfrequently in peptide synthesis. Accordingly, genes can be tailored foroptimal gene expression in a given organism based on codon optimization.Codon usage tables are readily available, for example, at the “CodonUsage Database” available at kazusa.orjp/codon/and these tables can beadapted in a number of ways. See Nakamura, Y., et al. “Codon usagetabulated from the international DNA sequence databases: status for theyear 2000” Nucl. Acids Res. 28:292 (2000). Computer algorithms for codonoptimizing a particular sequence for expression in a particular hostcell are also available, such as Gene Forge (Aptagen; Jacobus, Pa.), arealso available. In some embodiments, one or more codons (e.g. 1, 2, 3,4, 5, 10, 15, 20, 25, 50, or more, or all codons) in a sequence encodinga Cas correspond to the most frequently used codon for a particularamino acid.

In certain embodiments, the methods as described herein may compriseproviding a Cas transgenic cell, in particular a C2c2 transgenic cell,in which one or more nucleic acids encoding one or more guide RNAs areprovided or introduced operably connected in the cell with a regulatoryelement comprising a promoter of one or more gene of interest. As usedherein, the term “Cas transgenic cell” refers to a cell, such as aeukaryotic cell, in which a Cas gene has been genomically integrated.The nature, type, or origin of the cell are not particularly limitingaccording to the present invention. Also the way the Cas transgene isintroduced in the cell may vary and can be any method as is known in theart. In certain embodiments, the Cas transgenic cell is obtained byintroducing the Cas transgene in an isolated cell. In certain otherembodiments, the Cas transgenic cell is obtained by isolating cells froma Cas transgenic organism. By means of example, and without limitation,the Cas transgenic cell as referred to herein may be derived from a Castransgenic eukaryote, such as a Cas knock-in eukaryote. Reference ismade to WO 2014/093622 (PCT/US13/74667), incorporated herein byreference. Methods of US Patent Publication Nos. 20120017290 and20110265198 assigned to Sangamo BioSciences, Inc. directed to targetingthe Rosa locus may be modified to utilize the CRISPR Cas system of thepresent invention. Methods of US Patent Publication No. 20130236946assigned to Cellectis directed to targeting the Rosa locus may also bemodified to utilize the CRISPR Cas system of the present invention. Bymeans of further example reference is made to Platt et. al. (Cell;159(2):440-455 (2014)), describing a Cas9 knock-in mouse, which isincorporated herein by reference. The Cas transgene can further comprisea Lox-Stop-polyA-Lox(LSL) cassette thereby rendering Cas expressioninducible by Cre recombinase. Alternatively, the Cas transgenic cell maybe obtained by introducing the Cas transgene in an isolated cell.Delivery systems for transgenes are well known in the art. By means ofexample, the Cas transgene may be delivered in for instance eukaryoticcell by means of vector (e.g., AAV, adenovirus, lentivirus) and/orparticle and/or nanoparticle delivery, as also described hereinelsewhere.

It will be understood by the skilled person that the cell, such as theCas transgenic cell, as referred to herein may comprise further genomicalterations besides having an integrated Cas gene or the mutationsarising from the sequence specific action of Cas when complexed with RNAcapable of guiding Cas to a target locus.

In certain aspects the invention involves vectors, e.g. for deliveringor introducing in a cell Cas and/or RNA capable of guiding Cas to atarget locus (i.e. guide RNA), but also for propagating these components(e.g. in prokaryotic cells). A used herein, a “vector” is a tool thatallows or facilitates the transfer of an entity from one environment toanother. It is a replicon, such as a plasmid, phage, or cosmid, intowhich another DNA segment may be inserted so as to bring about thereplication of the inserted segment. Generally, a vector is capable ofreplication when associated with the proper control elements. Ingeneral, the term “vector” refers to a nucleic acid molecule capable oftransporting another nucleic acid to which it has been linked. Vectorsinclude, but are not limited to, nucleic acid molecules that aresingle-stranded, double-stranded, or partially double-stranded; nucleicacid molecules that comprise one or more free ends, no free ends (e.g.circular); nucleic acid molecules that comprise DNA, RNA, or both; andother varieties of polynucleotides known in the art. One type of vectoris a “plasmid,” which refers to a circular double stranded DNA loop intowhich additional DNA segments can be inserted, such as by standardmolecular cloning techniques. Another type of vector is a viral vector,wherein virally-derived DNA or RNA sequences are present in the vectorfor packaging into a virus (e.g. retroviruses, replication defectiveretroviruses, adenoviruses, replication defective adenoviruses, andadeno-associated viruses (AAVs)). Viral vectors also includepolynucleotides carried by a virus for transfection into a host cell.Certain vectors are capable of autonomous replication in a host cellinto which they are introduced (e.g. bacterial vectors having abacterial origin of replication and episomal mammalian vectors). Othervectors (e.g., non-episomal mammalian vectors) are integrated into thegenome of a host cell upon introduction into the host cell, and therebyare replicated along with the host genome. Moreover, certain vectors arecapable of directing the expression of genes to which they areoperatively-linked. Such vectors are referred to herein as “expressionvectors.” Common expression vectors of utility in recombinant DNAtechniques are often in the form of plasmids.

Recombinant expression vectors can comprise a nucleic acid of theinvention in a form suitable for expression of the nucleic acid in ahost cell, which means that the recombinant expression vectors includeone or more regulatory elements, which may be selected on the basis ofthe host cells to be used for expression, that is operatively-linked tothe nucleic acid sequence to be expressed. Within a recombinantexpression vector, “operably linked” is intended to mean that thenucleotide sequence of interest is linked to the regulatory element(s)in a manner that allows for expression of the nucleotide sequence (e.g.in an in vitro transcription/translation system or in a host cell whenthe vector is introduced into the host cell). With regards torecombination and cloning methods, mention is made of U.S. patentapplication Ser. No. 10/815,730, published Sep. 2, 2004 as US2004-0171156 A1, the contents of which are herein incorporated byreference in their entirety. Thus, the embodiments disclosed herein mayalso comprise transgenic cells comprising the CRISPR effector system. Incertain example embodiments, the transgenic cell may function as anindividual discrete volume. In other words samples comprising a maskingconstruct may be delivered to a cell, for example in a suitable deliveryvesicle and if the target is present in the delivery vesicle the CRISPReffector is activated and a detectable signal generated.

The vector(s) can include the regulatory element(s), e.g., promoter(s).The vector(s) can comprise Cas encoding sequences, and/or a single, butpossibly also can comprise at least 3 or 8 or 16 or 32 or 48 or 50 guideRNA(s) (e.g., sgRNAs) encoding sequences, such as 1-2, 1-3, 1-4 1-5,3-6, 3-7, 3-8, 3-9, 3-10, 3-8, 3-16, 3-30, 3-32, 3-48, 3-50 RNA(s)(e.g., sgRNAs). In a single vector there can be a promoter for each RNA(e.g., sgRNA), advantageously when there are up to about 16 RNA(s); and,when a single vector provides for more than 16 RNA(s), one or morepromoter(s) can drive expression of more than one of the RNA(s), e.g.,when there are 32 RNA(s), each promoter can drive expression of twoRNA(s), and when there are 48 RNA(s), each promoter can drive expressionof three RNA(s). By simple arithmetic and well established cloningprotocols and the teachings in this disclosure one skilled in the artcan readily practice the invention as to the RNA(s) for a suitableexemplary vector such as AAV, and a suitable promoter such as the U6promoter. For example, the packaging limit of AAV is ˜4.7 kb. The lengthof a single U6-gRNA (plus restriction sites for cloning) is 361 bp.Therefore, the skilled person can readily fit about 12-16, e.g., 13U6-gRNA cassettes in a single vector. This can be assembled by anysuitable means, such as a golden gate strategy used for TALE assembly(genome-engineering.org/taleffectors/). The skilled person can also usea tandem guide strategy to increase the number of U6-gRNAs byapproximately 1.5 times, e.g., to increase from 12-16, e.g., 13 toapproximately 18-24, e.g., about 19 U6-gRNAs. Therefore, one skilled inthe art can readily reach approximately 18-24, e.g., about 19promoter-RNAs, e.g., U6-gRNAs in a single vector, e.g., an AAV vector. Afurther means for increasing the number of promoters and RNAs in avector is to use a single promoter (e.g., U6) to express an array ofRNAs separated by cleavable sequences. And an even further means forincreasing the number of promoter-RNAs in a vector, is to express anarray of promoter-RNAs separated by cleavable sequences in the intron ofa coding sequence or gene; and, in this instance it is advantageous touse a polymerase II promoter, which can have increased expression andenable the transcription of long RNA in a tissue specific manner. (see,e.g., nar.oxfordjournals.org/content/34/7/e53.short andnature.com/mt/journal/v16/n9/abs/mt2008144a.html). In an advantageousembodiment, AAV may package U6 tandem gRNA targeting up to about 50genes. Accordingly, from the knowledge in the art and the teachings inthis disclosure the skilled person can readily make and use vector(s),e.g., a single vector, expressing multiple RNAs or guides under thecontrol or operatively or functionally linked to one or morepromoters—especially as to the numbers of RNAs or guides discussedherein, without any undue experimentation.

The guide RNA(s) encoding sequences and/or Cas encoding sequences, canbe functionally or operatively linked to regulatory element(s) and hencethe regulatory element(s) drive expression. The promoter(s) can beconstitutive promoter(s) and/or conditional promoter(s) and/or induciblepromoter(s) and/or tissue specific promoter(s). The promoter can beselected from the group consisting of RNA polymerases, pol I, pol II,pol III, T7, U6, H1, retroviral Rous sarcoma virus (RSV) LTR promoter,the cytomegalovirus (CMV) promoter, the SV40 promoter, the dihydrofolatereductase promoter, the β-actin promoter, the phosphoglycerol kinase(PGK) promoter, and the EF1α promoter. An advantageous promoter is thepromoter is U6.

In some embodiments, one or more elements of a nucleic acid-targetingsystem is derived from a particular organism comprising an endogenousCRISPR RNA-targeting system. In certain example embodiments, theeffector protein CRISPR RNA-targeting system comprises at least one HEPNdomain, including but not limited to the HEPN domains described herein,HEPN domains known in the art, and domains recognized to be HEPN domainsby comparison to consensus sequence motifs. Several such domains areprovided herein. In one non-limiting example, a consensus sequence canbe derived from the sequences of C2c2 or Cas13b orthologs providedherein. In certain example embodiments, the effector protein comprises asingle HEPN domain. In certain other example embodiments, the effectorprotein comprises two HEPN domains.

In one example embodiment, the effector protein comprise one or moreHEPN domains comprising a RxxxxH motif sequence. The RxxxxH motifsequence can be, without limitation, from a HEPN domain described hereinor a HEPN domain known in the art. RxxxxH motif sequences furtherinclude motif sequences created by combining portions of two or moreHEPN domains. As noted, consensus sequences can be derived from thesequences of the orthologs disclosed in U.S. Provisional PatentApplication 62/432,240 entitled “Novel CRISPR Enzymes and Systems,” U.S.Provisional Patent Application 62/471,710 entitled “Novel Type VI CRISPROrthologs and Systems” filed on Mar. 15, 2017, and U.S. Provisionalpatent application Ser. No. ______ entitled “Novel Type VI CRISPROrthologs and Systems,” labeled as attorney docket number 47627-05-2133and filed on Apr. 12, 2017.

In an embodiment of the invention, a HEPN domain comprises at least oneRxxxxH motif comprising the sequence of R{N/H/K}X1X2X3H. In anembodiment of the invention, a HEPN domain comprises a RxxxxH motifcomprising the sequence of R{N/H}X1X2X3H. In an embodiment of theinvention, a HEPN domain comprises the sequence of R{N/K}X1X2X3H. Incertain embodiments, X1 is R, S, D, E, Q, N, G, Y, or H. In certainembodiments, X2 is I, S, T, V, or L. In certain embodiments, X3 is L, F,N, Y, V, I, S, D, E, or A.

Additional effectors for use according to the invention can beidentified by their proximity to cas1 genes, for example, though notlimited to, within the region 20 kb from the start of the cas1 gene and20 kb from the end of the cas1 gene. In certain embodiments, theeffector protein comprises at least one HEPN domain and at least 500amino acids, and wherein the C2c2 effector protein is naturally presentin a prokaryotic genome within 20 kb upstream or downstream of a Casgene or a CRISPR array. Non-limiting examples of Cas proteins includeCas1, Cas1B, Cas2, Cas3, Cas4, Cas5, Cas6, Cas7, Cas8, Cas9 (also knownas Csn1 and Csx12), Cas10, Csy1, Csy2, Csy3, Cse1, Cse2, Csc1, Csc2,Csa5, Csn2, Csm2, Csm3, Csm4, Csm5, Csm6, Cmr1, Cmr3, Cmr4, Cmr5, Cmr6,Csb1, Csb2, Csb3, Csx17, Csx14, Csx10, Csx16, CsaX, Csx3, Csx1, Csx15,Csf1, Csf2, Csf3, Csf4, homologues thereof, or modified versionsthereof. In certain example embodiments, the C2c2 effector protein isnaturally present in a prokaryotic genome within 20 kb upstream ordownstream of a Cas 1 gene. The terms “orthologue” (also referred to as“ortholog” herein) and “homologue” (also referred to as “homolog”herein) are well known in the art. By means of further guidance, a“homologue” of a protein as used herein is a protein of the same specieswhich performs the same or a similar function as the protein it is ahomologue of. Homologous proteins may but need not be structurallyrelated, or are only partially structurally related. An “orthologue” ofa protein as used herein is a protein of a different species whichperforms the same or a similar function as the protein it is anorthologue of. Orthologous proteins may but need not be structurallyrelated, or are only partially structurally related.

In particular embodiments, the Type VI RNA-targeting Cas enzyme is C2c2.In other example embodiments, the Type VI RNA-targeting Cas enzyme isCas 13b. In particular embodiments, the homologue or orthologue of aType VI protein such as C2c2 as referred to herein has a sequencehomology or identity of at least 30%, or at least 40%, or at least 50%,or at least 60%, or at least 70%, or at least 80%, more preferably atleast 85%, even more preferably at least 90%, such as for instance atleast 95% with a Type VI protein such as C2c2 (e.g., based on thewild-type sequence of any of Leptotrichia shahii C2c2, Lachnospiraceaebacterium MA2020 C2c2, Lachnospiraceae bacterium NK4A179 C2c2,Clostridium aminophilum (DSM 10710) C2c2, Carnobacterium gallinarum (DSM4847) C2c2, Paludibacter propionicigenes (WB4) C2c2, Listeriaweihenstephanensis (FSL R9-0317) C2c2, Listeriaceae bacterium (FSLM6-0635) C2c2, Listeria newyorkensis (FSL M6-0635) C2c2, Leptotrichiawadei (F0279) C2c2, Rhodobacter capsulatus (SB 1003) C2c2, Rhodobactercapsulatus (R121) C2c2, Rhodobacter capsulatus (DE442) C2c2,Leptotrichia wadei (Lw2) C2c2, or Listeria seeligeri C2c2). In furtherembodiments, the homologue or orthologue of a Type VI protein such asC2c2 as referred to herein has a sequence identity of at least 30%, orat least 40%, or at least 50%, or at least 60%, or at least 70%, or atleast 80%, more preferably at least 85%, even more preferably at least90%, such as for instance at least 95% with the wild type C2c2 (e.g.,based on the wild-type sequence of any of Leptotrichia shahii C2c2,Lachnospiraceae bacterium MA2020 C2c2, Lachnospiraceae bacterium NK4A179C2c2, Clostridium aminophilum (DSM 10710) C2c2, Carnobacteriumgallinarum (DSM 4847) C2c2, Paludibacter propionicigenes (WB4) C2c2,Listeria weihenstephanensis (FSL R9-0317) C2c2, Listeriaceae bacterium(FSL M6-0635) C2c2, Listeria newyorkensis (FSL M6-0635) C2c2,Leptotrichia wadei (F0279) C2c2, Rhodobacter capsulatus (SB 1003) C2c2,Rhodobacter capsulatus (R121) C2c2, Rhodobacter capsulatus (DE442) C2c2,Leptotrichia wadei (Lw2) C2c2, or Listeria seeligeri C2c2).

In certain other example embodiments, the CRISPR system the effectorprotein is a C2c2 nuclease. The activity of C2c2 may depend on thepresence of two HEPN domains. These have been shown to be RNase domains,i.e. nuclease (in particular an endonuclease) cutting RNA. C2c2 HEPN mayalso target DNA, or potentially DNA and/or RNA. On the basis that theHEPN domains of C2c2 are at least capable of binding to and, in theirwild-type form, cutting RNA, then it is preferred that the C2c2 effectorprotein has RNase function. Regarding C2c2 CRISPR systems, reference ismade to U.S. Provisional 62/351,662 filed on Jun. 17, 2016 and U.S.Provisional 62/376,377 filed on Aug. 17, 2016. Reference is also made toU.S. Provisional 62/351,803 filed on Jun. 17, 2016. Reference is alsomade to U.S. Provisional entitled “Novel Crispr Enzymes and Systems”filed Dec. 8, 2016 bearing Broad Institute No. 10035.PA4 and AttorneyDocket No. 47627.03.2133. Reference is further made to East-Seletsky etal. “Two distinct RNase activities of CRISPR-C2c2 enable guide-RNAprocessing and RNA detection” Nature doi:10/1038/nature19802 andAbudayyeh et al. “C2c2 is a single-component programmable RNA-guided RNAtargeting CRISPR effector” bioRxiv doi:10.1101/054742.

RNase function in CRISPR systems is known, for example mRNA targetinghas been reported for certain type III CRISPR-Cas systems (Hale et al.,2014, Genes Dev, vol. 28, 2432-2443; Hale et al., 2009, Cell, vol. 139,945-956; Peng et al., 2015, Nucleic acids research, vol. 43, 406-417)and provides significant advantages. In the Staphylococcus epidermistype III-A system, transcription across targets results in cleavage ofthe target DNA and its transcripts, mediated by independent active siteswithin the Cas10-Csm ribonucleoprotein effector protein complex (see,Samai et al., 2015, Cell, vol. 151, 1164-1174). A CRISPR-Cas system,composition or method targeting RNA via the present effector proteins isthus provided.

In an embodiment, the Cas protein may be a C2c2 ortholog of an organismof a genus which includes but is not limited to Leptotrichia, Listeria,Corynebacter, Sutterella, Legionella, Treponema, Filifactor,Eubacterium, Streptococcus, Lactobacillus, Mycoplasma, Bacteroides,Flaviivola, Flavobacterium, Sphaerochaeta, Azospirillum,Gluconacetobacter, Neisseria, Roseburia, Parvibaculum, Staphylococcus,Nitratifractor, Mycoplasma and Campylobacter. Species of organism ofsuch a genus can be as otherwise herein discussed.

Some methods of identifying orthologues of CRISPR-Cas system enzymes mayinvolve identifying tracr sequences in genomes of interest.Identification of tracr sequences may relate to the following steps:Search for the direct repeats or tracr mate sequences in a database toidentify a CRISPR region comprising a CRISPR enzyme. Search forhomologous sequences in the CRISPR region flanking the CRISPR enzyme inboth the sense and antisense directions. Look for transcriptionalterminators and secondary structures. Identify any sequence that is nota direct repeat or a tracr mate sequence but has more than 50% identityto the direct repeat or tracr mate sequence as a potential tracrsequence. Take the potential tracr sequence and analyze fortranscriptional terminator sequences associated therewith.

It will be appreciated that any of the functionalities described hereinmay be engineered into CRISPR enzymes from other orthologs, includingchimeric enzymes comprising fragments from multiple orthologs. Examplesof such orthologs are described elsewhere herein. Thus, chimeric enzymesmay comprise fragments of CRISPR enzyme orthologs of an organism whichincludes but is not limited to Leptotrichia, Listeria, Corynebacter,Sutterella, Legionella, Treponema, Filifactor, Eubacterium,Streptococcus, Lactobacillus, Mycoplasma, Bacteroides, Flaviivola,Flavobacterium, Sphaerochaeta, Azospirillum, Gluconacetobacter,Neisseria, Roseburia, Parvibaculum, Staphylococcus, Nitratifractor,Mycoplasma and Campylobacter. A chimeric enzyme can comprise a firstfragment and a second fragment, and the fragments can be of CRISPRenzyme orthologs of organisms of genera herein mentioned or of speciesherein mentioned; advantageously the fragments are from CRISPR enzymeorthologs of different species.

In embodiments, the C2c2 protein as referred to herein also encompassesa functional variant of C2c2 or a homologue or an orthologue thereof. A“functional variant” of a protein as used herein refers to a variant ofsuch protein which retains at least partially the activity of thatprotein. Functional variants may include mutants (which may beinsertion, deletion, or replacement mutants), including polymorphs, etc.Also included within functional variants are fusion products of suchprotein with another, usually unrelated, nucleic acid, protein,polypeptide or peptide. Functional variants may be naturally occurringor may be man-made. Advantageous embodiments can involve engineered ornon-naturally occurring Type VI RNA-targeting effector protein.

In an embodiment, nucleic acid molecule(s) encoding the C2c2 or anortholog or homolog thereof, may be codon-optimized for expression in aeukaryotic cell. A eukaryote can be as herein discussed. Nucleic acidmolecule(s) can be engineered or non-naturally occurring.

In an embodiment, the C2c2 or an ortholog or homolog thereof, maycomprise one or more mutations (and hence nucleic acid molecule(s)coding for same may have mutation(s). The mutations may be artificiallyintroduced mutations and may include but are not limited to one or moremutations in a catalytic domain. Examples of catalytic domains withreference to a Cas9 enzyme may include but are not limited to RuvC I,RuvC II, RuvC III and HNH domains.

In an embodiment, the C2c2 or an ortholog or homolog thereof, maycomprise one or more mutations. The mutations may be artificiallyintroduced mutations and may include but are not limited to one or moremutations in a catalytic domain. Examples of catalytic domains withreference to a Cas enzyme may include but are not limited to HEPNdomains.

In an embodiment, the C2c2 or an ortholog or homolog thereof, may beused as a generic nucleic acid binding protein with fusion to or beingoperably linked to a functional domain. Exemplary functional domains mayinclude but are not limited to translational initiator, translationalactivator, translational repressor, nucleases, in particularribonucleases, a spliceosome, beads, a light inducible/controllabledomain or a chemically inducible/controllable domain.

In certain example embodiments, the C2c2 effector protein may be from anorganism selected from the group consisting of; Leptotrichia, Listeria,Corynebacter, Sutterella, Legionella, Treponema, Filifactor,Eubacterium, Streptococcus, Lactobacillus, Mycoplasma, Bacteroides,Flaviivola, Flavobacterium, Sphaerochaeta, Azospirillum,Gluconacetobacter, Neisseria, Roseburia, Parvibaculum, Staphylococcus,Nitratifractor, Mycoplasma, and Campylobacter.

In certain embodiments, the effector protein may be a Listeria sp.C2c2p, preferably Listeria seeligeria C2c2p, more preferably Listeriaseeligeria serovar 1/2b str. SLCC3954 C2c2p and the crRNA sequence maybe 44 to 47 nucleotides in length, with a 5′ 29-nt direct repeat (DR)and a 15-nt to 18-nt spacer.

In certain embodiments, the effector protein may be a Leptotrichia sp.C2c2p, preferably Leptotrichia shahii C2c2p, more preferablyLeptotrichia shahii DSM 19757 C2c2p and the crRNA sequence may be 42 to58 nucleotides in length, with a 5′ direct repeat of at least 24 nt,such as a 5′ 24-28-nt direct repeat (DR) and a spacer of at least 14 nt,such as a 14-nt to 28-nt spacer, or a spacer of at least 18 nt, such as19, 20, 21, 22, or more nt, such as 18-28, 19-28, 20-28, 21-28, or 22-28nt.

In certain example embodiments, the effector protein may be aLeptotrichia sp., Leptotrichia wadei F0279, or a Listeria sp.,preferably Listeria newyorkensis FSL M6-0635.

In certain example embodiments, the C2c2 effector proteins of theinvention include, without limitation, the following 21 ortholog species(including multiple CRISPR loci: Leptotrichia shahii; Leptotrichia wadei(Lw2); Listeria seeligeri; Lachnospiraceae bacterium MA2020;Lachnospiraceae bacterium NK4A179; [Clostridium] aminophilum DSM 10710;Carnobacterium gallinarum DSM 4847; Carnobacterium gallinarum DSM 4847(second CRISPR Loci); Paludibacter propionicigenes WB4; Listeriaweihenstephanensis FSL R9-0317; Listeriaceae bacterium FSL M6-0635;Leptotrichia wadei F0279; Rhodobacter capsulatus SB 1003; Rhodobactercapsulatus R121; Rhodobacter capsulatus DE442; Leptotrichia buccalisC-1013-b; Herbinix hemicellulosilytica; [Eubacterium] rectale;Eubacteriaceae bacterium CHKCI004; Blautia sp. Marseille-P2398; andLeptotrichia sp. oral taxon 879 str. F0557. Twelve (12) furthernon-limiting examples are: Lachnospiraceae bacterium NK4A144;Chloroflexus aggregans; Demequina aurantiaca; Thalassospira sp. TSLS-1;Pseudobutyrivibrio sp. OR37; Butyrivibrio sp. YAB3001; Blautia sp.Marseille-P2398; Leptotrichia sp. Marseille-P3007; Bacteroides ihuae;Porphyromonadaceae bacterium KH3CP3RA; Listeria riparia; andInsolitispirillum peregrinum.

In certain embodiments, the C2c2 protein according to the invention isor is derived from one of the orthologues as described in the tablebelow, or is a chimeric protein of two or more of the orthologues asdescribed in the table below, or is a mutant or variant of one of theorthologues as described in the table below (or a chimeric mutant orvariant), including dead C2c2, split C2c2, destabilized C2c2, etc. asdefined herein elsewhere, with or without fusion with aheterologous/functional domain.

In certain example embodiments, the C2c2 effector protein is selectedfrom Table 1 below.

TABLE 1 C2c2 orthologue Code Multi Letter Leptotrichia shahii C2-2 LshL. wadei F0279 (Lw2) C2-3 Lw2 Listeria seeligeri C2-4 LseLachnospiraceae bacterium MA2020 C2-5 LbM Lachnospiraceae bacteriumNK4A179 C2-6 LbNK179 Clostridium aminophilum DSM 10710 C2-7 CaCarnobacterium gallinarum DSM 4847 C2-8 Cg Carnobacterium gallinarum DSM4847 C2-9 Cg2 Paludibacter propionicigenes WB4 C2-10 Pp Listeriaweihenstephanensis FSL R9-0317 C2-11 Lwei Listeriaceae bacterium FSLM6-0635 C2-12 LbFSL Leptotrichia wadei F0279 C2-13 Lw Rhodobactercapsulatus SB 1003 C2-14 Rc Rhodobacter capsulatus R121 C2-15 RcRhodobacter capsulatus DE442 C2-16 Rc Leptotrichia buccalis C-1013-bC2-17 LbuC2c2 Herbinix hemicellulosilytics C2-18 HheC2c2 Eubacteriumrectale C2-19 EreC2c2 Eubacteriaceae bacterium CHKC1004 C2-20 EbaC2c2Blautia sp. Marseille-P2398 C2-21 BsmC2c2 Leptotrichia sp. oral taxon879 str. F0557 C2-22 LspC2c2 Lachnospiraceae bacterium NK4a144Chloroflexus aggregans Demequina aurantiaca Thalassospira sp. TSL5-1Pseudobutyrivibrio sp. OR37 Butyrivibrio sp. YAB3001 Blautia sp.Marseille-P2398 Leptotrichia sp. Marseille-P300 Bacteroides ihuaePorphyromonadaceae bacterium KH3CP3RA Listeria riparia Insolitispirillumperegrinum

The wild type protein sequences of the above species are listed in theTable 2 below. In certain embodiments, a nucleic acid sequence encodingthe C2c2 protein is provided.

TABLE 2 C2c2-2 L. shahii (Lsh) (SEQ. I.D. No. 1) C2c2-2 L. shahii (Lsh)(SEQ. I.D. No. 477) WP_018451595.1 c2c2-3 L. wadei (Lw2) (SEQ. I.D. No.2) c2c2-4 Listeria seeligeri (SEQ. I.D. No. 3) c2c2-5 1 Lachnospiraceaebacterium MA2020 (SEQ. I.D. No. 4) c2c2-6 2 Lachnospiraceae bacteriumNK4A179 (SEQ. I.D. No. 5) c2c2-7 3 Clostridium aminophilum DSM 10710(SEQ. I.D. No. 6) c2c2-8 5 Carnobacterium gallinarum DSM 4847 (SEQ. I.D.No. 7) c2c2-9 6 Carnobacterium gallinarum DSM 4847 (SEQ. I.D. No. 8)c2c2-10 7 Paludibacter propionicigenes WB4 (SEQ. I.D. No. 9) c2c2-11 9Listeria weihenstephanensis FSL R9-0317 (SEQ. I.D. No. 10) c2c2-12 10Listeriaceae bacterium FSL M6-0635 = Listeria newyorkensis FSL M6-0635(SEQ. I.D. No. 11) c2c2-13 12 Leptotrichia wadei F0279 (SEQ. I.D. No.12) c2c2-14 15 Rhodobacter capsulatus SB 1003 (SEQ. I.D. No. 13) c2c2-1516 Rhodobacter capsulatus R121 (SEQ. I.D. No. 14) c2c2-16 17 Rhodobactercapsulatus DE442 (SEQ. I.D. No. 15) LbuC2c2 (C2-17) Leptorichia buccalisC-1013-b (SEQ ID NO: 309) HheC2c2 (C2-18) Herbinix hemicellulosilytica(SEQ ID NO: 310) EreC2c2 (C2-19) Eubacterium rectale (SEQ ID NO: 311)EbaC2C2 (C2-20) Eubacteriaceae bacterium CHKCI004 (SEQ ID NO: 312) C2c2Blautia sp. Marseille-P2398 (SEQ. I.D. No 319 (C2-21) C2c2 Leptotrichiasp. Oral taxon 879 str. F0557 (C2-22) (SEQ. I.D. No. 579) C2c2 NK4A144Lachnospiraceae bacterium NK4A144 (SEQ. I.D. No. (C2-23) 313) C2c2Chloro_agg (C2-24) RNA-binding protein S1 Chloroflexus aggregans (SEQ.I.D. No. 314) C2c2 Dem_Aur (C2-25) Demequina aurantiaca (SEQ. I.D. No.315) C2c2 Thal_Sp_TSL5 (C2- Thalassospira sp. TSL5-1 (SEQ. I.D. No 316)26) C2c2 Pseudo_sp (C2-27) Pseudobutyrivibrio sp. OR37 (SEQ. I.D. No.317) C2c2_Buty_sp (C2-28) Butyrivibrio sp. YAB3001 (SEQ. I.D. No. 318)C2c2_Blautia_sp (C2-29) Blautia sp. Marseille-P2398(SEQ. I.D. No. 478)C2c2_Lepto_sp_Marseille Leptotrichia sp. Marseille-P3007 (SEQ. ID No.320) (C2-30) C2c2_Bacteroides_ihuae Bacteroides ihuae (SEQ. I.D. No 321)(C2-31) C2c2_Porph_bacterium Porphyromonadaceae bacterium KH3CP3RA(SEQ.I.D. (C2-32) No. 322) C2c2_Listeria_riparia (C2- Listeria riparia (SEQ.I.D. No. 323) 33) C2c2_insolitis_peregrinum Insolitispirillum peregrinum(SEQ. I.D. No. 324) (C2-34)

In an embodiment of the invention, there is provided effector proteinwhich comprises an amino acid sequence having at least 80% sequencehomology to the wild-type sequence of any of Leptotrichia shahii C2c2,Lachnospiraceae bacterium MA2020 C2c2, Lachnospiraceae bacterium NK4A179C2c2, Clostridium aminophilum (DSM 10710) C2c2, Carnobacteriumgallinarum (DSM 4847) C2c2, Paludibacter propionicigenes (WB4) C2c2,Listeria weihenstephanensis (FSL R9-0317) C2c2, Listeriaceae bacterium(FSL M6-0635) C2c2, Listeria newyorkensis (FSL M6-0635) C2c2,Leptotrichia wadei (F0279) C2c2, Rhodobacter capsulatus (SB 1003) C2c2,Rhodobacter capsulatus (R121) C2c2, Rhodobacter capsulatus (DE442) C2c2,Leptotrichia wadei (Lw2) C2c2, or Listeria seeligeri C2c2.

In an embodiment of the invention, the effector protein comprises anamino acid sequence having at least 80% sequence homology to a Type VIeffector protein consensus sequence including but not limited to aconsensus sequence described herein.

According to the invention, a consensus sequence can be generated frommultiple C2c2 orthologs, which can assist in locating conserved aminoacid residues, and motifs, including but not limited to catalyticresidues and HEPN motifs in C2c2 orthologs that mediate C2c2 function.One such consensus sequence, generated from the 33 orthologs mentionedabove using Geneious alignment is:

(SEQ ID NO: 325) MKISKVXXXVXKKXXXGKLXKXVNERNRXAKRLSNXLBKYIXXIDKIXKKEXXKKFXAXEEITLKLNQXXXBXLXKAXXDLRKDNXYSXJKKILHNEDINXEEXELLINDXLEKLXKIESXKYSYQKXXXNYXMSVQEHSKKSIXRIXESAKRNKEALDKFLKEYAXLDPRMEXLAKLRKLLELYFYEKNDXIXXEEEXNVXXEIKXLKENHPDFVEXXXNKENAELNXYAIEXKKJLKYYFPXKXAKNSNDKIFEKQELKKWIHQJENAVERILLXXGKVXYKLQXGYLAELWKIRINEIFIKYIXVGKAVAXFALRNXXKBENDILGGKIXKKLNGITSFXYEKIKAEEILQREXAVEVAFAANXLYAXDLXXIRXSILQFFGGASNWDXFLFFHFATSXISDKKWNAELIXXKKJGLVIREKLYSNNVAMFYSKDDLEKLLNXLXXFXLRASQVPSFKKVYVRXBFPQNLLKKFNDEKDDEAYSAXYYLLKEIYYNXFLPYFSANNXFFFXVKNLVLKANKDKFXXAFXDIREMNXGSPIEYLXXTQXNXXNEGRKKEEKEXDFIKELLQIFXKGFDDYLKNNXXFILKFIPEPTEXIEIXXELQAWYIVGKELNARKXNLLGXFXSYLKLLDDIELRALRNENIKYQSSNXEKEVLEXCLELIGLLSLDLNDYFBDEXDFAXYJGKXLDFEKKXMKDLAELXPYDQNDGENPIVNRNIXLAKKYGTLNLLEKJXDKVSEKEIKEYYELKKEIEEYXXKGEELHEEWXQXKNRVEXRDILEYXEELXGQIINYNXLXNKVLLYFQLGLHYLLLDILGRLVGYTGIWERDAXLYQIAAMYXNGLPEYIXXKKNDKYKDGQIVGXKINXFKXDKKXLYNAGLELFENXNEHKNIXIRNYIAHFNYLSKAESSLLXYSENLRXLFSYDRKLKNAVXKSLINILLRHGMVLKFKFGTDKKSVXIRSXKKIXHLKSIAKKLYYPEVXVSKEYCKLVKXLLKY K

In another non-limiting example, a sequence alignment tool to assistgeneration of a consensus sequence and identification of conservedresidues is the MUSCLE alignment tool (www.ebi.ac.uk/Tools/msa/muscle/).For example, using MUSCLE, the following amino acid locations conservedamong C2c2 orthologs can be identified in Leptotrichia wadei C2c2:K2;K5; V6; E301; L331; 1335; N341; G351; K352; E375; L392; L396; D403;F446; 1466; 1470; R474 (HEPN); H475; H479 (HEPN), E508; P556; L561;I595; Y596; F600; Y669; I673; F681; L685; Y761; L676; L779; Y782; L836;D847; Y863; L869; I872; K879; 1933; L954; 1958; R961; Y965; E970; R971;D972; R1046 (HEPN), H1051 (HEPN), Y1075; D1076; K1078; K1080; I1083;I1090.

An exemplary sequence alignment of HEPN domains showing highly conservedresidues is shown in FIG. 50.

In certain example embodiments, the RNA-targeting effector protein is aType VI-B effector protein, such as Cas13b and Group 29 or Group 30proteins. In certain example embodiments, the RNA-targeting effectorprotein comprises one or more HEPN domains. In certain exampleembodiments, the RNA-targeting effector protein comprises a C-terminalHEPN domain, a N-terminal HEPN domain, or both. Regarding example TypeVI-B effector proteins that may be used in the context of thisinvention, reference is made to U.S. application Ser. No. 15/331,792entitled “Novel CRISPR Enzymes and Systems” and filed Oct. 21, 2016,International Patent Application No. PCT/US2016/058302 entitled “NovelCRISPR Enzymes and Systems”, and filed Oct. 21, 2016, and Smargon et al.“Cas13b is a Type VI-B CRISPR-associated RNA-Guided RNase differentiallyregulated by accessory proteins Csx27 and Csx28” Molecular Cell, 65,1-13 (2017); dx.doi.org/10.1016/j.molcel.2016.12.023, and U.S.Provisional application Ser. No. ______, entitled “Novel Cas13bOrthologues CRISPR Enzymes and System” filed Mar. 15, 2017. Inparticular embodiments, the Cas13b enzyme is derived from Bergeyellazoohelcum. In certain other example embodiments, the effector proteinis, or comprises an amino acid sequence having at least 80% sequencehomology to any of the sequences listed in Table 3.

TABLE 3 B-01 Bergeyella zoohelcum B-02 Prevotella intermedia B-03Prevotella buccae B-04 Alistipes sp. ZOR0009 B-05 Prevotella sp. MA2016B-06 Riemerella anatipestifer B-07 Prevotella aurantiaca B-08 Prevotellasaccharolytica B-09 Prevotella intermedia B-10 Capnocytophaga canimorsusB-11 Porphyromonas gulae B-12 Prevotella sp. P5-125 B-13 Flavobacteriumbranchiophilum B-14 Porphyromonas gingivalis B-15 Prevotella intermedia

In certain example embodiments, the wild type sequence of the Cas13borthologue is found in Table 4a or 4b below.

TABLE 4a Bergeyella zoohelcum (SEQ. I.D. No. 326)  1 Prevotellaintermedia (SEQ. I.D. No. 327)  2 Prevotella buccae (SEQ. I.D. No. 328) 3 Porphyromonas gingivalis (SEQ. I.D. No. 329)  4 Bacteroides pyogenes(SEQ. I.D. No. 330)  5 Alistipes sp. ZOR0009 (SEQ. I.D. No. 331)  6Prevotella sp. MA2016 (SEQ. I.D. No. 332)  7a Prevotella sp. MA2016(SEQ. I.D. No. 333)  7b Riemerella anatipestifer (SEQ. I.D. No. 334)  8Prevotella aurantiaca (SEQ. I.D. No. 335)  9 Prevotella saccharolytica(SEQ. I.D. No. 336) 10 HMPREF9712_03108 [Myroides odoratimimus CCUG10230] (SEQ. I.D. No. 337) 11 Prevotella intermedia (SEQ. I.D. No. 338)12 Capnocytophaga canimorsus (SEQ. I.D. No. 339) 13 Porphyromonas gulae(SEQ. I.D. No. 340) 14 Prevotella sp. P5-125 (SEQ. I.D. No. 341) 15Flavobacterium branchiophilum (SEQ. I.D. No. 342) 16 Myroidesodoratimimus (SEQ. I.D. No. 343) 17 Flavobacterium columnare (SEQ. I.D.No. 344) 18 Porphyromonas gingivalis (SEQ. I.D. No. 345) 19Porphyromonas sp. COT-052 OH4946 (SEQ. I.D. No. 346) 20 Prevotellaintermedia (SEQ. I.D. No. 347) 21 PIN17_0200 [Prevotella intermedia 17](SEQ. I.D. No. 348) AFJ07523 Prevotella intermedia (SEQ. I.D. No. 349)BAU18623 HMPREF6485_0083 [Prevotella buccae ATCC 33574] (SEQ. I.D. No.350) EFU31981 HMPREF9144_1146 [Prevotella pallens ATCC 700821] (SEQ.I.D. No. 351) EGQ18444 HMPREF9714_02132 [Myroides odoratimimus CCUG12901] (SEQ. I.D. No. 352) EHO08761 HMPREF9711_00870 [Myroidesodoratimimus CCUG 3837] (SEQ. I.D. No. 353) EKB06014 HMPREF9699_02005[Bergeyella zoohelcum ATCC 43767] (SEQ. I.D. No. 354) EKB54193HMPREF9151_01387 [Prevotella saccharolytica F0055] (SEQ. I.D. No. 355)EKY00089 A343_1752 [Porphyromonas gingivalis JCVI SC001] (SEQ. I.D. No.356) EOA10535 HMPREF1981_03090 [Bacteroides pyogenes F0041] (SEQ. I.D.No. 357) ERI81700 HMPREF1553_02065 [Porphyromonas gingivalis F0568](SEQ. I.D. No. 358) ERJ65637 HMPREF1988_01768 [Porphyromonas gingivalisF0185] (SEQ. I.D. No. 359) ERJ81987 HMPREF1990_01800 [PorphyromonasgingivalisW4087] (SEQ. I.D. No. 360) ERJ87335 M573_117042 [Prevotellaintermedia ZT] (SEQ. I.D. No. 361) KJJ86756 A2033_10205 [Bacteroidetesbacterium GWA2_31_9] (SEQ. I.D. No. 362) OFX18020.1 SAMN05421542_0666[Chryseobacterium jejuense] (SEQ. I.D. No. 363) SDI27289.1SAMN05444360_11366 [Chryseobacterium carnipullorum] (SEQ. I.D. No. 364)SHM52812.1 SAMN05421786_1011119 [Chryseobacterium ureilyticum] (SEQ.I.D. No. 365) SIS70481.1 Prevotella buccae (SEQ. I.D. No. 366)WP_004343581 Porphyromonas gingivalis (SEQ. I.D. No. 367) WP_005873511Porphyromonas gingivalis (SEQ. I.D. No. 368) WP_005874195 Prevotellapallens (SEQ. I.D. No. 369) WP_006044833 Myroides odoratimimus (SEQ.I.D. No. 370) WP_006261414 Myroides odoratimimus (SEQ. I.D. No. 371)WP_006265509 Prevotella sp. MSX73 (SEQ. I.D. No. 372) WP_007412163Porphyromonas gingivalis (SEQ. I.D. No. 373) WP_012458414 Paludibacterpropionicigenes (SEQ. I.D. No. 374) WP_013446107 Porphyromonasgingivalis (SEQ. I.D. No. 375) WP_013816155 Flavobacterium columnare(SEQ. I.D. No. 376) WP_014165541 Psychroflexus torquis (SEQ. I.D. No.377) WP_015024765 Riemerella anatipestifer (SEQ. I.D. No. 378)WP_015345620 Prevotella pleuritidis (SEQ. I.D. No. 379) WP_021584635Porphyromonas gingivalis (SEQ. I.D. No. 380) WP_021663197 Porphyromonasgingivalis (SEQ. I.D. No. 381) WP_021665475 Porphyromonas gingivalis(SEQ. I.D. No. 382) WP_021677657 Porphyromonas gingivalis (SEQ. I.D. No.383) WP_021680012 Porphyromonas gingivalis (SEQ. I.D. No. 384)WP_023846767 Prevotella falsenii (SEQ. I.D. No. 385) WP_036884929Prevotella pleuritidis (SEQ. I.D. No. 386) WP_036931485 [Porphyromonasgingivalis (SEQ. I.D. No. 387) WP_039417390 Porphyromonas gulae (SEQ.I.D. No. 388) WP_039418912 Porphyromonas gulae (SEQ. I.D. No. 389)WP_039419792 Porphyromonas gulae (SEQ. I.D. No. 390) WP_039426176Porphyromonas gulae (SEQ. I.D. No. 391) WP_039431778 Porphyromonas gulae(SEQ. I.D. No. 392) WP_039437199 Porphyromonas gulae (SEQ. I.D. No. 393)WP_039442171 Porphyromonas gulae (SEQ. I.D. No. 394) WP_039445055Capnocytophaga cynodegmi (SEQ. I.D. No. 395) WP_041989581 Prevotella sp.P5-119 (SEQ. I.D. No. 396) WP_042518169 Prevotella sp. P4-76 (SEQ. I.D.No. 397) WP_044072147 Prevotella sp. P5-60 (SEQ. I.D. No. 398)WP_044074780 Phaeodactylibacter xiamenensis (SEQ. I.D. No. 399)WP_044218239 Flavobacterium sp. 316 (SEQ. I.D. No. 400) WP_045968377Porphyromonas gulae (SEQ. I.D. No. 401) WP_046201018 WP_047431796 (SEQ.I.D. No. 402) Chryseobacterium sp. YR477 Riemerella anatipestifer (SEQ.I.D. No. 403) WP_049354263 Porphyromonas gingivalis (SEQ. I.D. No. 404)WP_052912312 Porphyromonas gingivalis (SEQ. I.D. No. 405) WP_058019250Flavobacterium columnare (SEQ. I.D. No. 406) WP_060381855 Porphyromonasgingivalis (SEQ. I.D. No. 407) WP_061156470 Porphyromonas gingivalis(SEQ. I.D. No. 408) WP_061156637 Riemerella anatipestifer (SEQ. I.D. No.409) WP_061710138 Flavobacterium columnare (SEQ. I.D. No. 410)WP_063744070 Riemerella anatipestifer (SEQ. I.D. No. 411) WP_064970887Sinomicrobium oceani (SEQ. I.D. No. 412) WP_072319476.1 Reichenbachiellaagariperforans (SEQ. I.D. No. 413) WP_073124441.1

TABLE 4b Name or Accession No. WP_015345620 (SEQ. I.D. No. 479)WP_049354263 (SEQ. I.D. No. 480) WP_061710138 (SEQ. I.D. No. 481) 6(SEQ. I.D. No. 482) Alistipes sp. ZOR0009 SIS70481.1 15 Prevotella sp.(SEQ. I.D. No. 484) WP_042518169 (SEQ. I.D. No. 485) WP_044072147 (SEQ.I.D. No. 486) WP_044074780 (SEQ. I.D. No. 487) 8_(modified) (SEQ. I.D.No. 488) WP_064970887 (SEQ. I.D. No. 489) 5 (SEQ. I.D. No. 490) ERI81700(SEQ. I.D. No. 491) WP_036931485 (SEQ. I.D. No. 492) 19 (SEQ. I.D. No.493) WP_012458414 (SEQ. I.D. No. 494) WP_013816155 (SEQ. I.D. No. 495)WP_039417390 (SEQ. I.D. No. 496) WP_039419792 (SEQ. I.D. No. 497)WP_039426176 (SEQ. I.D. No. 498) WP_039437199 (SEQ. I.D. No. 499)WP_061156470 (SEQ. I.D. No. 500) 12 (SEQ. I.D. No. 501) 9 (SEQ. I.D. No.502) EGQ18444 (SEQ. I.D. No. 503) KJJ86756 (SEQ. I.D. No. 504)WP_006044833 (SEQ. I.D. No. 505) 2 (SEQ. I.D. No. 506) 3 (SEQ. I.D. No.507) EFU31981 (SEQ. I.D. No. 508) WP_004343581 (SEQ. I.D. No. 509)WP_007412163 (SEQ. I.D. No. 510) WP_044218239 (SEQ. I.D. No. 511) 21(SEQ. I.D. No. 512) BAU18623 (SEQ. I.D. No. 513) WP_036884929 (SEQ. I.D.No. 514) WP_073124441.1 (SEQ. I.D. No. 515) AFJ07523 (SEQ. I.D. No. 516)4 (SEQ. I.D. No. 517) ERJ65637 (SEQ. I.D. No. 518) ERJ81987 (SEQ. I.D.No. 519) ERJ87335 (SEQ. I.D. No. 520) WP_005873511 (SEQ. I.D. No. 521)WP_021663197 (SEQ. I.D. No. 522) WP_021665475 (SEQ. I.D. No. 523)WP_021677657 (SEQ. I.D. No. 524) WP_021680012 (SEQ. I.D. No. 525)WP_023846767 (SEQ. I.D. No. 526) WP_039445055 (SEQ. I.D. No. 527)WP_061156637 (SEQ. I.D. No. 528) WP_021584635 (SEQ. I.D. No. 529)WP_015024765 (SEQ. I.D. No. 530) WP_047431796 (SEQ. I.D. No. 531)WP_072319476.1 (SEQ. I.D. No. 532) 16 (SEQ. I.D. No. 533) EKY00089 (SEQ.I.D. No. 534) 10 (SEQ. I.D. No. 535) WP_013446107 (SEQ. I.D. No. 536)WP_045968377 (SEQ. I.D. No. 537) SHM52812.1 (SEQ. I.D. No. 538) EHO08761(SEQ. I.D. No. 539) EKB06014 (SEQ. I.D. No. 540) WP_006261414 (SEQ. I.D.No. 541) WP_006265509 (SEQ. I.D. No. 542) 11 (SEQ. I.D. No. 543) 17(SEQ. I.D. No. 544) OFX18020.1 (SEQ. I.D. No. 545) SDI27289.1 (SEQ. I.D.No. 546) WP_039442171 (SEQ. I.D. No. 547) 14 (SEQ. I.D. No. 548) 20(SEQ. I.D. No. 549) EOA10535 (SEQ. I.D. No. 550) WP_005874195 (SEQ. I.D.No. 551) WP_039418912 (SEQ. I.D. No. 552) WP_039431778 (SEQ. I.D. No.553) WP_046201018 (SEQ. I.D. No. 554) WP_052912312 (SEQ. I.D. No. 555)WP_058019250 (SEQ. I.D. No. 556) WP_014165541 (SEQ. I.D. No. 557) 13(SEQ. I.D. No. 558) WP_060381855 (SEQ. I.D. No. 559) WP_063744070 (SEQ.I.D. No. 560) 18 (SEQ. I.D. No. 561) WP_041989581 (SEQ. I.D. No. 562) 1(SEQ. I.D. No. 563) EKB54193 (SEQ. I.D. No. 564) 7_(modified) (SEQ. I.D.No. 565) 7_(modified)_-_residues_only (SEQ. I.D. No. 566)

In certain example embodiments, the RNA-targeting effector protein is aCas13c effector protein as disclosed in U.S. Provisional PatentApplication No. 62/525,165 filed Jun. 26, 2017, and PCT Application No.US 2017/047193 filed Aug. 16, 2017. Example wildtype orthologuesequences of Cas13c are provided in Table 5 below.

TABLE 5 Name EHO19081 (SEQ. I.D. No. 567) WP_094899336 (SEQ. I.D. No.568) WP_040490876 (SEQ. I.D. No. 569) WP_047396607 (SEQ. I.D. No. 570)WP_035935671 (SEQ. I.D. No. 571) WP_035906563 (SEQ. I.D. No. 572)WP_042678931 (SEQ. I.D. No. 573) WP_062627846 (SEQ. I.D. No. 574)WP_005959231 (SEQ. I.D. No. 575) WP_027128616 (SEQ. I.D. N. 576)WP_062624740 (SEQ. I.D. No. 577) WP_096402050 (SEQ. I.D. No. 578)

In certain example embodiments, the Cas13 protein may be selected fromany of the following.

TABLE 6 Seq. ID Species ID.No: Cas13a1 Leptotrichia shahii 580 Cas13a2Leptotrichia wadei (Lw2) 581 Cas13a3 Listeria seeligeri 582 Cas13a4Lachnospiraceae bacterium MA2020 583 Cas13a5 Lachnospiraceae bacteriumNK4A179 584 Cas13a6 [Clostridium] aminophilum DSM 10710 585 Cas13a7Carnobacterium gallinarum DSM 4847 586 Cas13a8 Carnobacterium gallinarumDSM 4847 587 Cas13a9 Paludibacter propionicigenes WB4 588 Cas13a10Listeria weihenstephanensis FSL R9-0317 589 Cas13a11 Listeriaceaebacterium FSL M6-0635 590 Cas13a12 Leptotrichia wadei F0279 591 Cas13a13Rhodobacter capsulatus SB 1003 592 Cas13a14 Rhodobacter capsulatus R121593 Cas13a15 Rhodobacter capsulatus DE442 594 Cas13a16 Leptotrichiabuccalis C-1013-b 595 Cas13a17 Herbinix hemicellulosilytica 596 Cas13a18[Eubacterium] rectale 597 Cas13a19 Eubacteriaceae bacterium CHKCI004 598Cas13a20 Blautia sp. Marseille-P2398 599 Cas13a21 Leptotrichia sp. oraltaxon 879 str. F0557 600 Cas13b1 Bergeyella zoohelcum 601 Cas13b2Prevotella intermedia 602 Cas13b3 Prevotella buccae 603 Cas13b4Alistipes sp. ZOR0009 604 Cas13b5 Prevotella sp. MA2016 605 Cas13b6Riemerella anatipestifer 606 Cas13b7 Prevotella aurantiaca 607 Cas13b8Prevotella saccharolytica 608 Cas13b9 Prevotella intermedia 609 Cas13b10Capnocytophaga canimorsus 610 Cas13b11 Porphyromonas gulae 611 Cas13b12Prevotella sp. P5-125 612 Cas13b13 Flavobacterium branchiophilum 613Cas13b14 Porphyromonas gingivalis 614 Cas13b15 Prevotella intermedia 615Cas13c1 Fusobacterium necrophorum subsp. funduliforme 616 ATCC 51357contig00003 Cas13c2 Fusobacterium necrophorum DJ-2 contig0065, 617 wholegenome shotgun sequence Cas13c3 Fusobacterium necrophorum BFTR-1contig0068 618 Ca13c4 Fusobacterium necrophorum subsp. funduliforme 6191_1_36S cont1.14 Cas13c5 Fusobacterium perfoetens ATCC 29250 620T364DRAFT_scaffold00009.9_C Cas13c6 Fusobacterium ulcerans ATCC 49185cont2.38 621 Cas13c7 Anaerosalibacter sp. ND1 genome assembly 622Anaerosalibacter massiliensis ND1

Guide Sequences

As used herein, the term “guide sequence,” “crRNA,” “guide RNA,” or“single guide RNA,” or “gRNA” refers to a polynucleotide comprising anypolynucleotide sequence having sufficient complementarity with a targetnucleic acid sequence to hybridize with the target nucleic acid sequenceand to direct sequence-specific binding of a RNA-targeting complexcomprising the guide sequence and a CRISPR effector protein to thetarget nucleic acid sequence. In some example embodiments, the degree ofcomplementarity, when optimally aligned using a suitable alignmentalgorithm, is about or more than about 50%, 60%, 75%, 80%, 85%, 90%,95%, 97.5%, 99%, or more. Optimal alignment may be determined with theuse of any suitable algorithm for aligning sequences, non-limitingexample of which include the Smith-Waterman algorithm, theNeedleman-Wunsch algorithm, algorithms based on the Burrows-WheelerTransform (e.g., the Burrows Wheeler Aligner), ClustalW, Clustal X,BLAT, Novoalign (Novocraft Technologies; available atwww.novocraft.com), ELAND (Illumina, San Diego, Calif.), SOAP (availableat soap.genomics.org.cn), and Maq (available at maq.sourceforge.net).The ability of a guide sequence (within a nucleic acid-targeting guideRNA) to direct sequence-specific binding of a nucleic acid-targetingcomplex to a target nucleic acid sequence may be assessed by anysuitable assay. For example, the components of a nucleic acid-targetingCRISPR system sufficient to form a nucleic acid-targeting complex,including the guide sequence to be tested, may be provided to a hostcell having the corresponding target nucleic acid sequence, such as bytransfection with vectors encoding the components of the nucleicacid-targeting complex, followed by an assessment of preferentialtargeting (e.g., cleavage) within the target nucleic acid sequence, suchas by Surveyor assay as described herein. Similarly, cleavage of atarget nucleic acid sequence may be evaluated in a test tube byproviding the target nucleic acid sequence, components of a nucleicacid-targeting complex, including the guide sequence to be tested and acontrol guide sequence different from the test guide sequence, andcomparing binding or rate of cleavage at the target sequence between thetest and control guide sequence reactions. Other assays are possible,and will occur to those skilled in the art. A guide sequence, and hencea nucleic acid-targeting guide may be selected to target any targetnucleic acid sequence. The target sequence may be DNA. The targetsequence may be any RNA sequence. In some embodiments, the targetsequence may be a sequence within a RNA molecule selected from the groupconsisting of messenger RNA (mRNA), pre-mRNA, ribosomal RNA (rRNA),transfer RNA (tRNA), micro-RNA (miRNA), small interfering RNA (siRNA),small nuclear RNA (snRNA), small nucleolar RNA (snoRNA), double strandedRNA (dsRNA), non-coding RNA (ncRNA), long non-coding RNA (lncRNA), andsmall cytoplasmatic RNA (scRNA). In some preferred embodiments, thetarget sequence may be a sequence within a RNA molecule selected fromthe group consisting of mRNA, pre-mRNA, and rRNA. In some preferredembodiments, the target sequence may be a sequence within a RNA moleculeselected from the group consisting of ncRNA, and lncRNA. In some morepreferred embodiments, the target sequence may be a sequence within anmRNA molecule or a pre-mRNA molecule.

In some embodiments, a nucleic acid-targeting guide is selected toreduce the degree secondary structure within the nucleic acid-targetingguide. In some embodiments, about or less than about 75%, 50%, 40%, 30%,25%, 20%, 15%, 10%, 5%, 1%, or fewer of the nucleotides of the nucleicacid-targeting guide participate in self-complementary base pairing whenoptimally folded. Optimal folding may be determined by any suitablepolynucleotide folding algorithm. Some programs are based on calculatingthe minimal Gibbs free energy. An example of one such algorithm ismFold, as described by Zuker and Stiegler (Nucleic Acids Res. 9 (1981),133-148). Another example folding algorithm is the online webserverRNAfold, developed at Institute for Theoretical Chemistry at theUniversity of Vienna, using the centroid structure prediction algorithm(see e.g., A. R. Gruber et al., 2008, Cell 106(1): 23-24; and PA Carrand GM Church, 2009, Nature Biotechnology 27(12): 1151-62).

In certain embodiments, a guide RNA or crRNA may comprise, consistessentially of, or consist of a direct repeat (DR) sequence and a guidesequence or spacer sequence. In certain embodiments, the guide RNA orcrRNA may comprise, consist essentially of, or consist of a directrepeat sequence fused or linked to a guide sequence or spacer sequence.In certain embodiments, the direct repeat sequence may be locatedupstream (i.e., 5′) from the guide sequence or spacer sequence. In otherembodiments, the direct repeat sequence may be located downstream (i.e.,3′) from the guide sequence or spacer sequence.

In certain embodiments, the crRNA comprises a stem loop, preferably asingle stem loop. In certain embodiments, the direct repeat sequenceforms a stem loop, preferably a single stem loop.

In certain embodiments, the spacer length of the guide RNA is from 15 to35 nt. In certain embodiments, the spacer length of the guide RNA is atleast 15 nucleotides. In certain embodiments, the spacer length is from15 to 17 nt, e.g., 15, 16, or 17 nt, from 17 to 20 nt, e.g., 17, 18, 19,or 20 nt, from 20 to 24 nt, e.g., 20, 21, 22, 23, or 24 nt, from 23 to25 nt, e.g., 23, 24, or 25 nt, from 24 to 27 nt, e.g., 24, 25, 26, or 27nt, from 27-30 nt, e.g., 27, 28, 29, or 30 nt, from 30-35 nt, e.g., 30,31, 32, 33, 34, or 35 nt, or 35 nt or longer.

In general, the CRISPR-Cas, CRISPR-Cas9 or CRISPR system may be as usedin the foregoing documents, such as WO 2014/093622 (PCT/US2013/074667)and refers collectively to transcripts and other elements involved inthe expression of or directing the activity of CRISPR-associated (“Cas”)genes, including sequences encoding a Cas gene, in particular a Cas9gene in the case of CRISPR-Cas9, a tracr (trans-activating CRISPR)sequence (e.g. tracrRNA or an active partial tracrRNA), a tracr-matesequence (encompassing a “direct repeat” and a tracrRNA-processedpartial direct repeat in the context of an endogenous CRISPR system), aguide sequence (also referred to as a “spacer” in the context of anendogenous CRISPR system), or “RNA(s)” as that term is herein used(e.g., RNA(s) to guide Cas9, e.g. CRISPR RNA and transactivating (tracr)RNA or a single guide RNA (sgRNA) (chimeric RNA)) or other sequences andtranscripts from a CRISPR locus. In general, a CRISPR system ischaracterized by elements that promote the formation of a CRISPR complexat the site of a target sequence (also referred to as a protospacer inthe context of an endogenous CRISPR system). In the context of formationof a CRISPR complex, “target sequence” refers to a sequence to which aguide sequence is designed to have complementarity, where hybridizationbetween a target sequence and a guide sequence promotes the formation ofa CRISPR complex. The section of the guide sequence through whichcomplementarity to the target sequence is important for cleavageactivity is referred to herein as the seed sequence. A target sequencemay comprise any polynucleotide, such as DNA or RNA polynucleotides. Insome embodiments, a target sequence is located in the nucleus orcytoplasm of a cell, and may include nucleic acids in or frommitochondrial, organelles, vesicles, liposomes or particles presentwithin the cell. In some embodiments, especially for non-nuclear uses,NLSs are not preferred. In some embodiments, a CRISPR system comprisesone or more nuclear exports signals (NESs). In some embodiments, aCRISPR system comprises one or more NLSs and one or more NESs. In someembodiments, direct repeats may be identified in silico by searching forrepetitive motifs that fulfill any or all of the following criteria: 1.found in a 2 Kb window of genomic sequence flanking the type II CRISPRlocus; 2. span from 20 to 50 bp; and 3. interspaced by 20 to 50 bp. Insome embodiments, 2 of these criteria may be used, for instance 1 and 2,2 and 3, or 1 and 3. In some embodiments, all 3 criteria may be used.

In embodiments of the invention the terms guide sequence and guide RNA,i.e. RNA capable of guiding Cas to a target genomic locus, are usedinterchangeably as in foregoing cited documents such as WO 2014/093622(PCT/US2013/074667). In general, a guide sequence is any polynucleotidesequence having sufficient complementarity with a target polynucleotidesequence to hybridize with the target sequence and directsequence-specific binding of a CRISPR complex to the target sequence. Insome embodiments, the degree of complementarity between a guide sequenceand its corresponding target sequence, when optimally aligned using asuitable alignment algorithm, is about or more than about 50%, 60%, 75%,80%, 85%, 90%, 95%, 97.5%, 99%, or more. Optimal alignment may bedetermined with the use of any suitable algorithm for aligningsequences, non-limiting example of which include the Smith-Watermanalgorithm, the Needleman-Wunsch algorithm, algorithms based on theBurrows-Wheeler Transform (e.g. the Burrows Wheeler Aligner), ClustalW,Clustal X, BLAT, Novoalign (Novocraft Technologies; available atwww.novocraft.com), ELAND (Illumina, San Diego, Calif.), SOAP (availableat soap.genomics.org.cn), and Maq (available at maq.sourceforge.net). Insome embodiments, a guide sequence is about or more than about 5, 10,11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28,29, 30, 35, 40, 45, 50, 75, or more nucleotides in length. In someembodiments, a guide sequence is less than about 75, 50, 45, 40, 35, 30,25, 20, 15, 12, or fewer nucleotides in length. Preferably the guidesequence is 10 30 nucleotides long. The ability of a guide sequence todirect sequence-specific binding of a CRISPR complex to a targetsequence may be assessed by any suitable assay. For example, thecomponents of a CRISPR system sufficient to form a CRISPR complex,including the guide sequence to be tested, may be provided to a hostcell having the corresponding target sequence, such as by transfectionwith vectors encoding the components of the CRISPR sequence, followed byan assessment of preferential cleavage within the target sequence, suchas by Surveyor assay as described herein. Similarly, cleavage of atarget polynucleotide sequence may be evaluated in a test tube byproviding the target sequence, components of a CRISPR complex, includingthe guide sequence to be tested and a control guide sequence differentfrom the test guide sequence, and comparing binding or rate of cleavageat the target sequence between the test and control guide sequencereactions. Other assays are possible, and will occur to those skilled inthe art.

In some embodiments of CRISPR-Cas systems, the degree of complementaritybetween a guide sequence and its corresponding target sequence can beabout or more than about 50%, 60%, 75%, 80%, 85%, 90%, 95%, 97.5%, 99%,or 100%; a guide or RNA or sgRNA can be about or more than about 5, 10,11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28,29, 30, 35, 40, 45, 50, 75, or more nucleotides in length; or guide orRNA or sgRNA can be less than about 75, 50, 45, 40, 35, 30, 25, 20, 15,12, or fewer nucleotides in length; and advantageously tracr RNA is 30or 50 nucleotides in length. However, an aspect of the invention is toreduce off-target interactions, e.g., reduce the guide interacting witha target sequence having low complementarity. Indeed, in the examples,it is shown that the invention involves mutations that result in theCRISPR-Cas system being able to distinguish between target andoff-target sequences that have greater than 80% to about 95%complementarity, e.g., 83%-84% or 88-89% or 94-95% complementarity (forinstance, distinguishing between a target having 18 nucleotides from anoff-target of 18 nucleotides having 1, 2 or 3 mismatches). Accordingly,in the context of the present invention the degree of complementaritybetween a guide sequence and its corresponding target sequence isgreater than 94.5% or 95% or 95.5% or 96% or 96.5% or 97% or 97.5% or98% or 98.5% or 99% or 99.5% or 99.9%, or 100%. Off target is less than100% or 99.9% or 99.5% or 99% or 99% or 98.5% or 98% or 97.5% or 97% or96.5% or 96% or 95.5% or 95% or 94.5% or 94% or 93% or 92% or 91% or 90%or 89% or 88% or 87% or 86% or 85% or 84% or 83% or 82% or 81% or 80%complementarity between the sequence and the guide, with it advantageousthat off target is 100% or 99.9% or 99.5% or 99% or 99% or 98.5% or 98%or 97.5% or 97% or 96.5% or 96% or 95.5% or 95% or 94.5% complementaritybetween the sequence and the guide.

Guide Modifications

In certain embodiments, guides of the invention comprise non-naturallyoccurring nucleic acids and/or non-naturally occurring nucleotidesand/or nucleotide analogs, and/or chemical modifications. Non-naturallyoccurring nucleic acids can include, for example, mixtures of naturallyand non-naturally occurring nucleotides. Non-naturally occurringnucleotides and/or nucleotide analogs may be modified at the ribose,phosphate, and/or base moiety. In an embodiment of the invention, aguide nucleic acid comprises ribonucleotides and non-ribonucleotides. Inone such embodiment, a guide comprises one or more ribonucleotides andone or more deoxyribonucleotides. In an embodiment of the invention, theguide comprises one or more non-naturally occurring nucleotide ornucleotide analog such as a nucleotide with phosphorothioate linkage,boranophosphate linkage, a locked nucleic acid (LNA) nucleotidescomprising a methylene bridge between the 2′ and 4′ carbons of theribose ring, or bridged nucleic acids (BNA). Other examples of modifiednucleotides include 2′-O-methyl analogs, 2′-deoxy analogs, 2-thiouridineanalogs, N6-methyladenosine analogs, or 2′-fluoro analogs. Furtherexamples of modified bases include, but are not limited to,2-aminopurine, 5-bromo-uridine, pseudouridine (Ψ),N¹-methylpseudouridine (me¹Ψ), 5-methoxyuridine(5moU), inosine,7-methylguanosine. Examples of guide RNA chemical modifications include,without limitation, incorporation of 2′-O-methyl (M),2′-O-methyl-3′-phosphorothioate (MS), phosphorothioate (PS),S-constrained ethyl(cEt), or 2′-O-methyl-3′-thioPACE (MSP) at one ormore terminal nucleotides. Such chemically modified guides can compriseincreased stability and increased activity as compared to unmodifiedguides, though on-target vs. off-target specificity is not predictable.(See, Hendel, 2015, Nat Biotechnol. 33(9):985-9, doi: 10.1038/nbt.3290,published online 29 Jun. 2015; Ragdarm et al., 0215, PNAS, E7110-E7111;Allerson et al., J. Med. Chem. 2005, 48:901-904; Bramsen et al., Front.Genet., 2012, 3:154; Deng et al., PNAS, 2015, 112:11870-11875; Sharma etal., Med Chem Comm., 2014, 5:1454-1471; Hendel et al., Nat. Biotechnol.(2015) 33(9): 985-989; Li et al., Nature Biomedical Engineering, 2017,1, 0066 DOI:10.1038/s41551-017-0066). In some embodiments, the 5′ and/or3′ end of a guide RNA is modified by a variety of functional moietiesincluding fluorescent dyes, polyethylene glycol, cholesterol, proteins,or detection tags. (See Kelly et al., 2016, J. Biotech. 233:74-83). Incertain embodiments, a guide comprises ribonucleotides in a region thatbinds to a target DNA and one or more deoxyribonucleotides and/ornucleotide analogs in a region that binds to Cas9, Cpf1, or C2c1. In anembodiment of the invention, deoxyribonucleotides and/or nucleotideanalogs are incorporated in engineered guide structures, such as,without limitation, 5′ and/or 3′ end, stem-loop regions, and the seedregion. In certain embodiments, the modification is not in the 5′-handleof the stem-loop regions. Chemical modification in the 5′-handle of thestem-loop region of a guide may abolish its function (see Li, et al.,Nature Biomedical Engineering, 2017, 1:0066). In certain embodiments, atleast 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19,20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 40, 45, 50, or 75nucleotides of a guide is chemically modified. In some embodiments, 3-5nucleotides at either the 3′ or the 5′ end of a guide is chemicallymodified. In some embodiments, only minor modifications are introducedin the seed region, such as 2′-F modifications. In some embodiments,2′-F modification is introduced at the 3′ end of a guide. In certainembodiments, three to five nucleotides at the 5′ and/or the 3′ end ofthe guide are chemically modified with 2′-O-methyl (M),2′-O-methyl-3′-phosphorothioate (MS), S-constrained ethyl(cEt), or2′-O-methyl-3′-thioPACE (MSP). Such modification can enhance genomeediting efficiency (see Hendel et al., Nat. Biotechnol. (2015) 33(9):985-989). In certain embodiments, all of the phosphodiester bonds of aguide are substituted with phosphorothioates (PS) for enhancing levelsof gene disruption. In certain embodiments, more than five nucleotidesat the 5′ and/or the 3′ end of the guide are chemically modified with2′-0-Me, 2′-F or S-constrained ethyl(cEt). Such chemically modifiedguide can mediate enhanced levels of gene disruption (see Ragdarm etal., 0215, PNAS, E7110-E7111). In an embodiment of the invention, aguide is modified to comprise a chemical moiety at its 3′ and/or 5′ end.Such moieties include, but are not limited to amine, azide, alkyne,thio, dibenzocyclooctyne (DBCO), or Rhodamine. In certain embodiment,the chemical moiety is conjugated to the guide by a linker, such as analkyl chain. In certain embodiments, the chemical moiety of the modifiedguide can be used to attach the guide to another molecule, such as DNA,RNA, protein, or nanoparticles. Such chemically modified guide can beused to identify or enrich cells generically edited by a CRISPR system(see Lee et al., eLife, 2017, 6:e25312, DOI:10.7554).

In certain embodiments, the CRISPR system as provided herein can makeuse of a crRNA or analogous polynucleotide comprising a guide sequence,wherein the polynucleotide is an RNA, a DNA or a mixture of RNA and DNA,and/or wherein the polynucleotide comprises one or more nucleotideanalogs. The sequence can comprise any structure, including but notlimited to a structure of a native crRNA, such as a bulge, a hairpin ora stem loop structure. In certain embodiments, the polynucleotidecomprising the guide sequence forms a duplex with a secondpolynucleotide sequence which can be an RNA or a DNA sequence.

In certain embodiments, use is made of chemically modified guide RNAs.Examples of guide RNA chemical modifications include, withoutlimitation, incorporation of 2′-O-methyl (M), 2′-O-methyl3′phosphorothioate (MS), or 2′-O-methyl 3′thioPACE (MSP) at one or moreterminal nucleotides. Such chemically modified guide RNAs can compriseincreased stability and increased activity as compared to unmodifiedguide RNAs, though on-target vs. off-target specificity is notpredictable. (See, Hendel, 2015, Nat Biotechnol. 33(9):985-9, doi:10.1038/nbt.3290, published online 29 Jun. 2015). Chemically modifiedguide RNAs further include, without limitation, RNAs withphosphorothioate linkages and locked nucleic acid (LNA) nucleotidescomprising a methylene bridge between the 2′ and 4′ carbons of theribose ring.

In some embodiments, a guide sequence is about or more than about 5, 10,11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28,29, 30, 35, 40, 45, 50, 75, or more nucleotides in length. In someembodiments, a guide sequence is less than about 75, 50, 45, 40, 35, 30,25, 20, 15, 12, or fewer nucleotides in length. Preferably the guidesequence is 10 to 30 nucleotides long. The ability of a guide sequenceto direct sequence-specific binding of a CRISPR complex to a targetsequence may be assessed by any suitable assay. For example, thecomponents of a CRISPR system sufficient to form a CRISPR complex,including the guide sequence to be tested, may be provided to a hostcell having the corresponding target sequence, such as by transfectionwith vectors encoding the components of the CRISPR sequence, followed byan assessment of preferential cleavage within the target sequence, suchas by Surveyor assay. Similarly, cleavage of a target RNA may beevaluated in a test tube by providing the target sequence, components ofa CRISPR complex, including the guide sequence to be tested and acontrol guide sequence different from the test guide sequence, andcomparing binding or rate of cleavage at the target sequence between thetest and control guide sequence reactions. Other assays are possible,and will occur to those skilled in the art.

In some embodiments, the modification to the guide is a chemicalmodification, an insertion, a deletion or a split. In some embodiments,the chemical modification includes, but is not limited to, incorporationof 2′-O-methyl (M) analogs, 2′-deoxy analogs, 2-thiouridine analogs,N6-methyladenosine analogs, 2′-fluoro analogs, 2-aminopurine,5-bromo-uridine, pseudouridine (Ψ), N¹-methylpseudouridine (me¹Ψ),5-methoxyuridine(5moU), inosine, 7-methylguanosine,2′-O-methyl-3′-phosphorothioate (MS), S-constrained ethyl(cEt),phosphorothioate (PS), or 2′-O-methyl-3′-thioPACE (MSP). In someembodiments, the guide comprises one or more of phosphorothioatemodifications. In certain embodiments, at least 1, 2, 3, 4, 5, 6, 7, 8,9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or 25 nucleotides of theguide are chemically modified. In certain embodiments, one or morenucleotides in the seed region are chemically modified. In certainembodiments, one or more nucleotides in the 3′-terminus are chemicallymodified. In certain embodiments, none of the nucleotides in the5′-handle is chemically modified. In some embodiments, the chemicalmodification in the seed region is a minor modification, such asincorporation of a 2′-fluoro analog. In a specific embodiment, onenucleotide of the seed region is replaced with a 2′-fluoro analog. Insome embodiments, 5 or 10 nucleotides in the 3′-terminus are chemicallymodified. Such chemical modifications at the 3′-terminus of the Cpf1CrRNA improve gene cutting efficiency (see Li, et al., Nature BiomedicalEngineering, 2017, 1:0066). In a specific embodiment, 5 nucleotides inthe 3′-terminus are replaced with 2′-fluoro analogues. In a specificembodiment, 10 nucleotides in the 3′-terminus are replaced with2′-fluoro analogues. In a specific embodiment, 5 nucleotides in the3′-terminus are replaced with 2′-O-methyl (M) analogs.

In some embodiments, the loop of the 5′-handle of the guide is modified.In some embodiments, the loop of the 5′-handle of the guide is modifiedto have a deletion, an insertion, a split, or chemical modifications. Incertain embodiments, the loop comprises 3, 4, or 5 nucleotides. Incertain embodiments, the loop comprises the sequence of UCUU, UUUU,UAUU, or UGUU.

A guide sequence, and hence a nucleic acid-targeting guide RNA may beselected to target any target nucleic acid sequence. In the context offormation of a CRISPR complex, “target sequence” refers to a sequence towhich a guide sequence is designed to have complementarity, wherehybridization between a target sequence and a guide sequence promotesthe formation of a CRISPR complex. A target sequence may comprise RNApolynucleotides. The term “target RNA” refers to a RNA polynucleotidebeing or comprising the target sequence. In other words, the target RNAmay be a RNA polynucleotide or a part of a RNA polynucleotide to which apart of the gRNA, i.e. the guide sequence, is designed to havecomplementarity and to which the effector function mediated by thecomplex comprising CRISPR effector protein and a gRNA is to be directed.In some embodiments, a target sequence is located in the nucleus orcytoplasm of a cell. The target sequence may be DNA. The target sequencemay be any RNA sequence. In some embodiments, the target sequence may bea sequence within a RNA molecule selected from the group consisting ofmessenger RNA (mRNA), pre-mRNA, ribosomal RNA (rRNA), transfer RNA(tRNA), micro-RNA (miRNA), small interfering RNA (siRNA), small nuclearRNA (snRNA), small nuclear RNA (snoRNA), double stranded RNA (dsRNA),non coding RNA (ncRNA), long non-coding RNA (lncRNA), and smallcytoplasmic RNA (scRNA). In some preferred embodiments, the targetsequence may be a sequence within a RNA molecule selected from the groupconsisting of mRNA, pre-mRNA, and rRNA. In some preferred embodiments,the target sequence may be a sequence within a RNA molecule selectedfrom the group consisting of ncRNA, and lncRNA. In some more preferredembodiments, the target sequence may be a sequence within an mRNAmolecule or a pre-mRNA molecule.

In certain embodiments, the spacer length of the guide RNA is less than28 nucleotides. In certain embodiments, the spacer length of the guideRNA is at least 18 nucleotides and less than 28 nucleotides. In certainembodiments, the spacer length of the guide RNA is between 19 and 28nucleotides. In certain embodiments, the spacer length of the guide RNAis between 19 and 25 nucleotides. In certain embodiments, the spacerlength of the guide RNA is 20 nucleotides. In certain embodiments, thespacer length of the guide RNA is 23 nucleotides. In certainembodiments, the spacer length of the guide RNA is 25 nucleotides.

In certain embodiments, modulations of cleavage efficiency can beexploited by introduction of mismatches, e.g. 1 or more mismatches, suchas 1 or 2 mismatches between spacer sequence and target sequence,including the position of the mismatch along the spacer/target. The morecentral (i.e. not 3′ or 5′) for instance a double mismatch is, the morecleavage efficiency is affected. Accordingly, by choosing mismatchposition along the spacer, cleavage efficiency can be modulated. Bymeans of example, if less than 100% cleavage of targets is desired (e.g.in a cell population), 1 or more, such as preferably 2 mismatchesbetween spacer and target sequence may be introduced in the spacersequences. The more central along the spacer of the mismatch position,the lower the cleavage percentage.

In certain example embodiments, the cleavage efficiency may be exploitedto design single guides that can distinguish two or more targets thatvary by a single nucleotide, such as a single nucleotide polymorphism(SNP), variation, or (point) mutation. The CRISPR effector may havereduced sensitivity to SNPs (or other single nucleotide variations) andcontinue to cleave SNP targets with a certain level of efficiency. Thus,for two targets, or a set of targets, a guide RNA may be designed with anucleotide sequence that is complementary to one of the targets i.e. theon-target SNP. The guide RNA is further designed to have a syntheticmismatch. As used herein a “synthetic mismatch” refers to anon-naturally occurring mismatch that is introduced upstream ordownstream of the naturally occurring SNP, such as at most 5 nucleotidesupstream or downstream, for instance 4, 3, 2, or 1 nucleotide upstreamor downstream, preferably at most 3 nucleotides upstream or downstream,more preferably at most 2 nucleotides upstream or downstream, mostpreferably 1 nucleotide upstream or downstream (i.e. adjacent the SNP).When the CRISPR effector binds to the on-target SNP, only a singlemismatch will be formed with the synthetic mismatch and the CRISPReffector will continue to be activated and a detectable signal produced.When the guide RNA hybridizes to an off-target SNP, two mismatches willbe formed, the mismatch from the SNP and the synthetic mismatch, and nodetectable signal generated. Thus, the systems disclosed herein may bedesigned to distinguish SNPs within a population. For, example thesystems may be used to distinguish pathogenic strains that differ by asingle SNP or detect certain disease specific SNPs, such as but notlimited to, disease associated SNPs, such as without limitation cancerassociated SNPs.

In certain embodiments, the guide RNA is designed such that the SNP islocated on position 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15,16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 of thespacer sequence (starting at the 5′ end). In certain embodiments, theguide RNA is designed such that the SNP is located on position 1, 2, 3,4, 5, 6, 7, 8, or 9 of the spacer sequence (starting at the 5′ end). Incertain embodiments, the guide RNA is designed such that the SNP islocated on position 2, 3, 4, 5, 6, or 7 of the spacer sequence (startingat the 5′ end). In certain embodiments, the guide RNA is designed suchthat the SNP is located on position 3, 4, 5, or 6 of the spacer sequence(starting at the 5′ end). In certain embodiments, the guide RNA isdesigned such that the SNP is located on position 3 of the spacersequence (starting at the 5′ end).

In certain embodiments, the guide RNA is designed such that the mismatch(e.g. the synthetic mismatch, i.e. an additional mutation besides a SNP)is located on position 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14,15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 of thespacer sequence (starting at the 5′ end). In certain embodiments, theguide RNA is designed such that the mismatch is located on position 1,2, 3, 4, 5, 6, 7, 8, or 9 of the spacer sequence (starting at the 5′end). In certain embodiments, the guide RNA is designed such that themismatch is located on position 4, 5, 6, or 7 of the spacer sequence(starting at the 5′ end. In certain embodiments, the guide RNA isdesigned such that the mismatch is located on position 5 of the spacersequence (starting at the 5′ end).

In certain embodiments, the guide RNA is designed such that the mismatchis located 2 nucleotides upstream of the SNP (i.e. one interveningnucleotide).

In certain embodiments, the guide RNA is designed such that the mismatchis located 2 nucleotides downstream of the SNP (i.e. one interveningnucleotide).

In certain embodiments, the guide RNA is designed such that the mismatchis located on position 5 of the spacer sequence (starting at the 5′ end)and the SNP is located on position 3 of the spacer sequence (starting atthe 5′ end).

The embodiments described herein comprehend inducing one or morenucleotide modifications in a eukaryotic cell (in vitro, i.e. in anisolated eukaryotic cell) as herein discussed comprising delivering tocell a vector as herein discussed. The mutation(s) can include theintroduction, deletion, or substitution of one or more nucleotides ateach target sequence of cell(s) via the guide(s) RNA(s). The mutationscan include the introduction, deletion, or substitution of 1-75nucleotides at each target sequence of said cell(s) via the guide(s)RNA(s). The mutations can include the introduction, deletion, orsubstitution of 1, 5, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21,22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 40, 45, 50, or 75 nucleotides ateach target sequence of said cell(s) via the guide(s) RNA(s). Themutations can include the introduction, deletion, or substitution of 5,10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27,28, 29, 30, 35, 40, 45, 50, or 75 nucleotides at each target sequence ofsaid cell(s) via the guide(s) RNA(s). The mutations include theintroduction, deletion, or substitution of 10, 11, 12, 13, 14, 15, 16,17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 40, 45, 50,or 75 nucleotides at each target sequence of said cell(s) via theguide(s) RNA(s). The mutations can include the introduction, deletion,or substitution of 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 40,45, 50, or 75 nucleotides at each target sequence of said cell(s) viathe guide(s) RNA(s). The mutations can include the introduction,deletion, or substitution of 40, 45, 50, 75, 100, 200, 300, 400 or 500nucleotides at each target sequence of said cell(s) via the guide(s)RNA(s).

Typically, in the context of an endogenous CRISPR system, formation of aCRISPR complex (comprising a guide sequence hybridized to a targetsequence and complexed with one or more Cas proteins) results incleavage in or near (e.g. within 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 50,or more base pairs from) the target sequence, but may depend on forinstance secondary structure, in particular in the case of RNA targets.

RNA-Based Masking Constructs

As used herein, a “masking construct” refers to a molecule that can becleaved or otherwise deactivated by an activated CRISPR system effectorprotein described herein. The term “masking construct” may also bereferred to in the alternative as a “detection construct.” In certainexample embodiments, the masking construct is a RNA-based maskingconstruct. The RNA-based masking construct comprises a RNA element thatis cleavable by a CRISPR effector protein. Cleavage of the RNA elementreleases agents or produces conformational changes that allow adetectable signal to be produced. Example constructs demonstrating howthe RNA element may be used to prevent or mask generation of detectablesignal are described below and embodiments of the invention comprisevariants of the same. Prior to cleavage, or when the masking constructis in an ‘active’ state, the masking construct blocks the generation ordetection of a positive detectable signal. It will be understood that incertain example embodiments a minimal background signal may be producedin the presence of an active RNA masking construct. A positivedetectable signal may be any signal that can be detected using optical,fluorescent, chemiluminescent, electrochemical or other detectionmethods known in the art. The term “positive detectable signal” is usedto differentiate from other detectable signals that may be detectable inthe presence of the masking construct. For example, in certainembodiments a first signal may be detected when the masking agent ispresent (i.e. a negative detectable signal), which then converts to asecond signal (e.g. the positive detectable signal) upon detection ofthe target molecules and cleavage or deactivation of the masking agentby the activated CRISPR effector protein.

In certain example embodiments, the masking construct may suppressgeneration of a gene product. The gene product may be encoded by areporter construct that is added to the sample. The masking constructmay be an interfering RNA involved in a RNA interference pathway, suchas a short hairpin RNA (shRNA) or small interfering RNA (siRNA). Themasking construct may also comprise microRNA (miRNA). While present, themasking construct suppresses expression of the gene product. The geneproduct may be a fluorescent protein or other RNA transcript or proteinsthat would otherwise be detectable by a labeled probe, aptamer, orantibody but for the presence of the masking construct. Upon activationof the effector protein the masking construct is cleaved or otherwisesilenced allowing for expression and detection of the gene product asthe positive detectable signal.

In certain example embodiments, the masking construct may sequester oneor more reagents needed to generate a detectable positive signal suchthat release of the one or more reagents from the masking constructresults in generation of the detectable positive signal. The one or morereagents may combine to produce a colorimetric signal, achemiluminescent signal, a fluorescent signal, or any other detectablesignal and may comprise any reagents known to be suitable for suchpurposes. In certain example embodiments, the one or more reagents aresequestered by RNA aptamers that bind the one or more reagents. The oneor more reagents are released when the effector protein is activatedupon detection of a target molecule and the RNA aptamers are degraded.

In certain example embodiments, the masking construct may be immobilizedon a solid substrate in an individual discrete volume (defined furtherbelow) and sequesters a single reagent. For example, the reagent may bea bead comprising a dye. When sequestered by the immobilized reagent,the individual beads are too diffuse to generate a detectable signal,but upon release from the masking construct are able to generate adetectable signal, for example by aggregation or simple increase insolution concentration. In certain example embodiments, the immobilizedmasking agent is a RNA-based aptamer that can be cleaved by theactivated effector protein upon detection of a target molecule.

In certain other example embodiments, the masking construct binds to animmobilized reagent in solution thereby blocking the ability of thereagent to bind to a separate labeled binding partner that is free insolution. Thus, upon application of a washing step to a sample, thelabeled binding partner can be washed out of the sample in the absenceof a target molecule. However, if the effector protein is activated, themasking construct is cleaved to a degree sufficient to interfere withthe ability of the masking construct to bind the reagent therebyallowing the labeled binding partner to bind to the immobilized reagent.Thus, the labeled binding partner remains after the wash step indicatingthe presence of the target molecule in the sample. In certain aspects,the masking construct that binds the immobilized reagent is a RNAaptamer. The immobilized reagent may be a protein and the labeledminding partner may be a labeled antibody. Alternatively, theimmobilized reagent may be streptavidin and the labeled binding partnermay be labeled biotin. The label on the binding partner used in theabove embodiments may be any detectable label known in the art. Inaddition, other known binding partners may be used in accordance withthe overall design described herein.

In certain example embodiments, the masking construct may comprise aribozyme. Ribozymes are RNA molecules having catalytic properties.Ribozymes, both naturally and engineered, comprise or consist of RNAthat may be targeted by the effector proteins disclosed herein. Theribozyme may be selected or engineered to catalyze a reaction thateither generates a negative detectable signal or prevents generation ofa positive control signal. Upon deactivation of the ribozyme by theactivated effector protein the reaction generating a negative controlsignal, or preventing generation of a positive detectable signal, isremoved thereby allowing a positive detectable signal to be generated.In one example embodiment, the ribozyme may catalyze a colorimetricreaction causing a solution to appear as a first color. When theribozyme is deactivated the solution then turns to a second color, thesecond color being the detectable positive signal. An example of howribozymes can be used to catalyze a colorimetric reaction are describedin Zhao et al. “Signal amplification of glucosamine-6-phosphate based onribozyme glmS,” Biosens Bioelectron. 2014; 16:337-42, and provide anexample of how such a system could be modified to work in the context ofthe embodiments disclosed herein. Alternatively, ribozymes, when presentcan generate cleavage products of, for example, RNA transcripts. Thus,detection of a positive detectable signal may comprise detection ofnon-cleaved RNA transcripts that are only generated in the absence ofthe ribozyme.

In certain example embodiments, the one or more reagents is a protein,such as an enzyme, capable of facilitating generation of a detectablesignal, such as a colorimetric, chemiluminescent, or fluorescent signal,that is inhibited or sequestered such that the protein cannot generatethe detectable signal by the binding of one or more RNA aptamers to theprotein. Upon activation of the effector proteins disclosed herein, theRNA aptamers are cleaved or degraded to an extent that they no longerinhibit the protein's ability to generate the detectable signal. Incertain example embodiments, the aptamer is a thrombin inhibitoraptamer. In certain example embodiments the thrombin inhibitor aptamerhas a sequence of GGGAACAAAGCUGAAGUACUUACCC (SEQ ID NO: 414). When thisaptamer is cleaved, thrombin will become active and will cleave apeptide colorimetric or fluorescent substrate. In certain exampleembodiments, the colorimetric substrate is para-nitroanilide (pNA)covalently linked to the peptide substrate for thrombin. Upon cleavageby thrombin, pNA is released and becomes yellow in color and easilyvisible to the eye. In certain example embodiments, the fluorescentsubstrate is 7-amino-4-methylcoumarin a blue fluorophore that can bedetected using a fluorescence detector. Inhibitory aptamers may also beused for horseradish peroxidase (HRP), beta-galactosidase, or calfalkaline phosphatase (CAP) and within the general principals laid outabove.

In certain embodiments, RNAse activity is detected colorimetrically viacleavage of enzyme-inhibiting aptamers. One potential mode of convertingRNAse activity into a colorimetric signal is to couple the cleavage ofan RNA aptamer with the re-activation of an enzyme that is capable ofproducing a colorimetric output. In the absence of RNA cleavage, theintact aptamer will bind to the enzyme target and inhibit its activity.The advantage of this readout system is that the enzyme provides anadditional amplification step: once liberated from an aptamer viacollateral activity (e.g. Cas13a collateral activity), the colorimetricenzyme will continue to produce colorimetric product, leading to amultiplication of signal.

In certain embodiments, an existing aptamer that inhibits an enzyme witha colorimetric readout is used. Several aptamer/enzyme pairs withcolorimetric readouts exist, such as thrombin, protein C, neutrophilelastase, and subtilisin. These proteases have colorimetric substratesbased upon pNA and are commercially available. In certain embodiments, anovel aptamer targeting a common colorimetric enzyme is used. Common androbust enzymes, such as beta-galactosidase, horseradish peroxidase, orcalf intestinal alkaline phosphatase, could be targeted by engineeredaptamers designed by selection strategies such as SELEX. Such strategiesallow for quick selection of aptamers with nanomolar bindingefficiencies and could be used for the development of additionalenzyme/aptamer pairs for colorimetric readout.

In certain embodiments, RNAse activity is detected colorimetrically viacleavage of RNA-tethered inhibitors. Many common colorimetric enzymeshave competitive, reversible inhibitors: for example, beta-galactosidasecan be inhibited by galactose. Many of these inhibitors are weak, buttheir effect can be increased by increases in local concentration. Bylinking local concentration of inhibitors to RNAse activity,colorimetric enzyme and inhibitor pairs can be engineered into RNAsesensors. The colorimetric RNAse sensor based upon small-moleculeinhibitors involves three components: the colorimetric enzyme, theinhibitor, and a bridging RNA that is covalently linked to both theinhibitor and enzyme, tethering the inhibitor to the enzyme. In theuncleaved configuration, the enzyme is inhibited by the increased localconcentration of the small molecule; when the RNA is cleaved (e.g. byCas13a collateral cleavage), the inhibitor will be released and thecolorimetric enzyme will be activated.

In certain embodiments, RNAse activity is detected colorimetrically viaformation and/or activation of G-quadruplexes. G quadraplexes in DNA cancomplex with heme (iron (III)-protoporphyrin IX) to form a DNAzyme withperoxidase activity. When supplied with a peroxidase substrate (e.g.ABTS: (2,2′-Azinobis [3-ethylbenzothiazoline-6-sulfonic acid]-diammoniumsalt)), the G-quadraplex-heme complex in the presence of hydrogenperoxide causes oxidation of the substrate, which then forms a greencolor in solution. An example G-quadraplex forming DNA sequence is:GGGTAGGGCGGGTTGGGA (SEQ. I.D. No. 415).

By hybridizing an RNA sequence to this DNA aptamer, formation of theG-quadraplex structure will be limited. Upon RNAse collateral activation(e.g. C2c2-complex collateral activation), the RNA staple will becleaved allowing the G quadraplex to form and heme to bind. Thisstrategy is particularly appealing because color formation is enzymatic,meaning there is additional amplification beyond RNAse activation.

In certain example embodiments, the masking construct may be immobilizedon a solid substrate in an individual discrete volume (defined furtherbelow) and sequesters a single reagent. For example, the reagent may bea bead comprising a dye. When sequestered by the immobilized reagent,the individual beads are too diffuse to generate a detectable signal,but upon release from the masking construct are able to generate adetectable signal, for example by aggregation or simple increase insolution concentration. In certain example embodiments, the immobilizedmasking agent is a RNA-based aptamer that can be cleaved by theactivated effector protein upon detection of a target molecule.

In one example embodiment, the masking construct comprises a detectionagent that changes color depending on whether the detection agent isaggregated or dispersed in solution. For example, certain nanoparticles,such as colloidal gold, undergo a visible purple to red color shift asthey move from aggregates to dispersed particles. Accordingly, incertain example embodiments, such detection agents may be held inaggregate by one or more bridge molecules. See e.g. FIG. 43. At least aportion of the bridge molecule comprises RNA. Upon activation of theeffector proteins disclosed herein, the RNA portion of the bridgemolecule is cleaved allowing the detection agent to disperse andresulting in the corresponding change in color. See e.g. FIG. 46. Incertain example embodiments the, bridge molecule is a RNA molecule. Incertain example embodiments, the detection agent is a colloidal metal.The colloidal metal material may include water-insoluble metal particlesor metallic compounds dispersed in a liquid, a hydrosol, or a metal sol.The colloidal metal may be selected from the metals in groups IA, IB,IIB and IIIB of the periodic table, as well as the transition metals,especially those of group VIII. Preferred metals include gold, silver,aluminum, ruthenium, zinc, iron, nickel and calcium. Other suitablemetals also include the following in all of their various oxidationstates: lithium, sodium, magnesium, potassium, scandium, titanium,vanadium, chromium, manganese, cobalt, copper, gallium, strontium,niobium, molybdenum, palladium, indium, tin, tungsten, rhenium,platinum, and gadolinium. The metals are preferably provided in ionicform, derived from an appropriate metal compound, for example the A13+,Ru3+, Zn2+, Fe3+, Ni2+ and Ca2+ ions.

When the RNA bridge is cut by the activated CRISPR effector, thebeforementioned color shift is observed. In certain example embodimentsthe particles are colloidal metals. In certain other exampleembodiments, the colloidal metal is a colloidal gold. In certain exampleembodiments, the colloidal nanoparticles are 15 nm gold nanoparticles(AuNPs). Due to the unique surface properties of colloidal goldnanoparticles, maximal absorbance is observed at 520 nm when fullydispersed in solution and appear red in color to the naked eye. Uponaggregation of AuNPs, they exhibit a red-shift in maximal absorbance andappear darker in color, eventually precipitating from solution as a darkpurple aggregate. In certain example embodiments the nanoparticles aremodified to include DNA linkers extending from the surface of thenanoparticle. Individual particles are linked together bysingle-stranded RNA (ssRNA) bridges that hybridize on each end of theRNA to at least a portion of the DNA linkers. Thus, the nanoparticleswill form a web of linked particles and aggregate, appearing as a darkprecipitate. Upon activation of the CRISPR effectors disclosed herein,the ssRNA bridge will be cleaved, releasing the AU NPS from the linkedmesh and producing a visible red color. Example DNA linkers and RNAbridge sequences are listed below. Thiol linkers on the end of the DNAlinkers may be used for surface conjugation to the AuNPS. Other forms ofconjugation may be used. In certain example embodiments, two populationsof AuNPs may be generated, one for each DNA linker. This will helpfacilitate proper binding of the ssRNA bridge with proper orientation.In certain example embodiments, a first DNA linker is conjugated by the3′ end while a second DNA linker is conjugated by the 5′ end.

C2c2 colorimetric TTATAACTATTCCTAAAAAAAAAAA/ DNA1 3ThioMC3-D/(SEQ. I.D. No. 183) C2c2 colorimetric /5ThioMC6- DNA2D/AAAAAAAAAACTCCCCTAATAACAAT (SEQ. I.D. No. 184) C2c2 colorimetricGGGUAGGAAUAGUUAUAAUUUCCCUUUCCCA bridge UUGUUAUUAGGGAG (SEQ. I.D.No. 185)

In certain other example embodiments, the masking construct may comprisean RNA oligonucleotide to which are attached a detectable label and amasking agent of that detectable label. An example of such a detectablelabel/masking agent pair is a fluorophore and a quencher of thefluorophore. Quenching of the fluorophore can occur as a result of theformation of a non-fluorescent complex between the fluorophore andanother fluorophore or non-fluorescent molecule. This mechanism is knownas ground-state complex formation, static quenching, or contactquenching. Accordingly, the RNA oligonucleotide may be designed so thatthe fluorophore and quencher are in sufficient proximity for contactquenching to occur. Fluorophores and their cognate quenchers are knownin the art and can be selected for this purpose by one having ordinaryskill in the art. The particular fluorophore/quencher pair is notcritical in the context of this invention, only that selection of thefluorophore/quencher pairs ensures masking of the fluorophore. Uponactivation of the effector proteins disclosed herein, the RNAoligonucleotide is cleaved thereby severing the proximity between thefluorophore and quencher needed to maintain the contact quenchingeffect. Accordingly, detection of the fluorophore may be used todetermine the presence of a target molecule in a sample.

In certain other example embodiments, the masking construct may compriseone or more RNA oligonucleotides to which are attached one or more metalnanoparticles, such as gold nanoparticles. In some embodiments, themasking construct comprises a plurality of metal nanoparticlescrosslinked by a plurality of RNA oligonucleotides forming a closedloop. In one embodiment, the masking construct comprises three goldnanoparticles crosslinked by three RNA oligonucleotides forming a closedloop. In some embodiments, the cleavage of the RNA oligonucleotides bythe CRISPR effector protein leads to a detectable signal produced by themetal nanoparticles.

In certain other example embodiments, the masking construct may compriseone or more RNA oligonucleotides to which are attached one or morequantum dots. In some embodiments, the cleavage of the RNAoligonucleotides by the CRISPR effector protein leads to a detectablesignal produced by the quantum dots.

In one example embodiment, the masking construct may comprise a quantumdot. The quantum dot may have multiple linker molecules attached to thesurface. At least a portion of the linker molecule comprises RNA. Thelinker molecule is attached to the quantum dot at one end and to one ormore quenchers along the length or at terminal ends of the linker suchthat the quenchers are maintained in sufficient proximity for quenchingof the quantum dot to occur. The linker may be branched. As above, thequantum dot/quencher pair is not critical, only that selection of thequantum dot/quencher pair ensures masking of the fluorophore. Quantumdots and their cognate quenchers are known in the art and can beselected for this purpose by one having ordinary skill in the art Uponactivation of the effector proteins disclosed herein, the RNA portion ofthe linker molecule is cleaved thereby eliminating the proximity betweenthe quantum dot and one or more quenchers needed to maintain thequenching effect. In certain example embodiments the quantum dot isstreptavidin conjugated. RNA are attached via biotin linkers and recruitquenching molecules with the sequences /5Biosg/UCUCGUACGUUC/3IAbRQSp/(SEQ ID NO. 416) or /5Biosg/UCUCGUACGUUCUCUCGUACGUUC/3IAbRQSp/ (SEQ IDNO. 417), where /5Biosg/ is a biotin tag and /31AbRQSp/ is an Iowa blackquencher. Upon cleavage, by the activated effectors disclosed herein thequantum dot will fluoresce visibly.

In a similar fashion, fluorescence energy transfer (FRET) may be used togenerate a detectable positive signal. FRET is a non-radiative processby which a photon from an energetically excited fluorophore (i.e. “donorfluorophore”) raises the energy state of an electron in another molecule(i.e. “the acceptor”) to higher vibrational levels of the excitedsinglet state. The donor fluorophore returns to the ground state withoutemitting a fluoresce characteristic of that fluorophore. The acceptorcan be another fluorophore or non-fluorescent molecule. If the acceptoris a fluorophore, the transferred energy is emitted as fluorescencecharacteristic of that fluorophore. If the acceptor is a non-fluorescentmolecule the absorbed energy is loss as heat. Thus, in the context ofthe embodiments disclosed herein, the fluorophore/quencher pair isreplaced with a donor fluorophore/acceptor pair attached to theoligonucleotide molecule. When intact, the masking construct generates afirst signal (negative detectable signal) as detected by thefluorescence or heat emitted from the acceptor. Upon activation of theeffector proteins disclosed herein the RNA oligonucleotide is cleavedand FRET is disrupted such that fluorescence of the donor fluorophore isnow detected (positive detectable signal).

In certain example embodiments, the masking construct comprises the useof intercalating dyes which change their absorbance in response tocleavage of long RNAs to short nucleotides. Several such dyes exist. Forexample, pyronine-Y will complex with RNA and form a complex that has anabsorbance at 572 nm. Cleavage of the RNA results in loss of absorbanceand a color change. Methylene blue may be used in a similar fashion,with changes in absorbance at 688 nm upon RNA cleavage. Accordingly, incertain example embodiments the masking construct comprises a RNA andintercalating dye complex that changes absorbance upon the cleavage ofRNA by the effector proteins disclosed herein.

In certain example embodiments, the masking construct may comprise aninitiator for an HCR reaction. See e.g. Dirks and Pierce. PNAS 101,15275-15728 (2004). HCR reactions utilize the potential energy in twohairpin species. When a single-stranded initiator having a portion ofcomplementary to a corresponding region on one of the hairpins isreleased into the previously stable mixture, it opens a hairpin of onespeces. This process, in turn, exposes a single-stranded region thatopens a hairpin of the other species. This process, in turn, exposes asingle stranded region identical to the original initiator. Theresulting chain reaction may lead to the formation of a nicked doublehelix that grows until the hairpin supply is exhausted. Detection of theresulting products may be done on a gel or colorimetrically. Examplecolorimetric detection methods include, for example, those disclosed inLu et al. “Ultra-sensitive colorimetric assay system based on thehybridization chain reaction-triggered enzyme cascade amplification ACSAppl Mater Interfaces, 2017, 9(1):167-175, Wang et al. “An enzyme-freecolorimetric assay using hybridization chain reaction amplification andsplit aptamers” Analyst 2015, 150, 7657-7662, and Song et al. “Noncovalent fluorescent labeling of hairpin DNA probe coupled withhybridization chain reaction for sensitive DNA detection.” AppliedSpectroscopy, 70(4): 686-694 (2016).

In certain example embodiments, the masking construct may comprise a HCRinitiator sequence and a cleavable structural element, such as a loop orhairpin, that prevents the initiator from initiating the HCR reaction.Upon cleavage of the structure element by an activated CRISPR effectorprotein, the initiator is then released to trigger the HCR reaction,detection thereof indicating the presence of one or more targets in thesample. In certain example embodiments, the masking construct comprisesa hairpin with a RNA loop. When an activated CRISRP effector proteincuts the RNA loop, the initiator can be released to trigger the HCRreaction.

Amplification of Target

In certain example embodiments, target RNAs and/or DNAs may be amplifiedprior to activating the CRISPR effector protein. Any suitable RNA or DNAamplification technique may be used. In certain example embodiments, theRNA or DNA amplification is an isothermal amplification. In certainexample embodiments, the isothermal amplification may be nucleic-acidsequenced-based amplification (NASBA), recombinase polymeraseamplification (RPA), loop-mediated isothermal amplification (LAMP),strand displacement amplification (SDA), helicase-dependentamplification (HDA), or nicking enzyme amplification reaction (NEAR). Incertain example embodiments, non-isothermal amplification methods may beused which include, but are not limited to, PCR, multiple displacementamplification (MDA), rolling circle amplification (RCA), ligase chainreaction (LCR), or ramification amplification method (RAM).

In certain example embodiments, the RNA or DNA amplification is NASBA,which is initiated with reverse transcription of target RNA by asequence-specific reverse primer to create a RNA/DNA duplex. RNase H isthen used to degrade the RNA template, allowing a forward primercontaining a promoter, such as the T7 promoter, to bind and initiateelongation of the complementary strand, generating a double-stranded DNAproduct. The RNA polymerase promoter-mediated transcription of the DNAtemplate then creates copies of the target RNA sequence. Importantly,each of the new target RNAs can be detected by the guide RNAs thusfurther enhancing the sensitivity of the assay. Binding of the targetRNAs by the guide RNAs then leads to activation of the CRISPR effectorprotein and the methods proceed as outlined above. The NASBA reactionhas the additional advantage of being able to proceed under moderateisothermal conditions, for example at approximately 41° C., making itsuitable for systems and devices deployed for early and direct detectionin the field and far from clinical laboratories.

In certain other example embodiments, a recombinase polymeraseamplification (RPA) reaction may be used to amplify the target nucleicacids. RPA reactions employ recombinases which are capable of pairingsequence-specific primers with homologous sequence in duplex DNA. Iftarget DNA is present, DNA amplification is initiated and no othersample manipulation such as thermal cycling or chemical melting isrequired. The entire RPA amplification system is stable as a driedformulation and can be transported safely without refrigeration. RPAreactions may also be carried out at isothermal temperatures with anoptimum reaction temperature of 37-42° C. The sequence specific primersare designed to amplify a sequence comprising the target nucleic acidsequence to be detected. In certain example embodiments, a RNApolymerase promoter, such as a T7 promoter, is added to one of theprimers. This results in an amplified double-stranded DNA productcomprising the target sequence and a RNA polymerase promoter. After, orduring, the RPA reaction, a RNA polymerase is added that will produceRNA from the double-stranded DNA templates. The amplified target RNA canthen in turn be detected by the CRISPR effector system. In this waytarget DNA can be detected using the embodiments disclosed herein. RPAreactions can also be used to amplify target RNA. The target RNA isfirst converted to cDNA using a reverse transcriptase, followed bysecond strand DNA synthesis, at which point the RPA reaction proceeds asoutlined above.

Accordingly, in certain example embodiments the systems disclosed hereinmay include amplification reagents. Different components or reagentsuseful for amplification of nucleic acids are described herein. Forexample, an amplification reagent as described herein may include abuffer, such as a Tris buffer. A Tris buffer may be used at anyconcentration appropriate for the desired application or use, forexample including, but not limited to, a concentration of 1 mM, 2 mM, 3mM, 4 mM, 5 mM, 6 mM, 7 mM, 8 mM, 9 mM, 10 mM, 11 mM, 12 mM, 13 mM, 14mM, 15 mM, 25 mM, 50 mM, 75 mM, 1 M, or the like. One of skill in theart will be able to determine an appropriate concentration of a buffersuch as Tris for use with the present invention.

A salt, such as magnesium chloride (MgCl₂), potassium chloride (KCl), orsodium chloride (NaCl), may be included in an amplification reaction,such as PCR, in order to improve the amplification of nucleic acidfragments. Although the salt concentration will depend on the particularreaction and application, in some embodiments, nucleic acid fragments ofa particular size may produce optimum results at particular saltconcentrations. Larger products may require altered salt concentrations,typically lower salt, in order to produce desired results, whileamplification of smaller products may produce better results at highersalt concentrations. One of skill in the art will understand that thepresence and/or concentration of a salt, along with alteration of saltconcentrations, may alter the stringency of a biological or chemicalreaction, and therefore any salt may be used that provides theappropriate conditions for a reaction of the present invention and asdescribed herein.

Other components of a biological or chemical reaction may include a celllysis component in order to break open or lyse a cell for analysis ofthe materials therein. A cell lysis component may include, but is notlimited to, a detergent, a salt as described above, such as NaCl, KCl,ammonium sulfate [(NH₄)₂SO₄], or others. Detergents that may beappropriate for the invention may include Triton X-100, sodium dodecylsulfate (SDS), CHAPS(3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate), ethyltrimethyl ammonium bromide, nonyl phenoxypolyethoxylethanol (NP-40).Concentrations of detergents may depend on the particular application,and may be specific to the reaction in some cases. Amplificationreactions may include dNTPs and nucleic acid primers used at anyconcentration appropriate for the invention, such as including, but notlimited to, a concentration of 100 nM, 150 nM, 200 nM, 250 nM, 300 nM,350 nM, 400 nM, 450 nM, 500 nM, 550 nM, 600 nM, 650 nM, 700 nM, 750 nM,800 nM, 850 nM, 900 nM, 950 nM, 1 mM, 2 mM, 3 mM, 4 mM, 5 mM, 6 mM, 7mM, 8 mM, 9 mM, 10 mM, 20 mM, 30 mM, 40 mM, 50 mM, 60 mM, 70 mM, 80 mM,90 mM, 100 mM, 150 mM, 200 mM, 250 mM, 300 mM, 350 mM, 400 mM, 450 mM,500 mM, or the like. Likewise, a polymerase useful in accordance withthe invention may be any specific or general polymerase known in the artand useful or the invention, including Taq polymerase, Q5 polymerase, orthe like.

In some embodiments, amplification reagents as described herein may beappropriate for use in hot-start amplification. Hot start amplificationmay be beneficial in some embodiments to reduce or eliminatedimerization of adaptor molecules or oligos, or to otherwise preventunwanted amplification products or artifacts and obtain optimumamplification of the desired product. Many components described hereinfor use in amplification may also be used in hot-start amplification. Insome embodiments, reagents or components appropriate for use withhot-start amplification may be used in place of one or more of thecomposition components as appropriate. For example, a polymerase orother reagent may be used that exhibits a desired activity at aparticular temperature or other reaction condition. In some embodiments,reagents may be used that are designed or optimized for use in hot-startamplification, for example, a polymerase may be activated aftertransposition or after reaching a particular temperature. Suchpolymerases may be antibody-based or aptamer-based. Polymerases asdescribed herein are known in the art. Examples of such reagents mayinclude, but are not limited to, hot-start polymerases, hot-start dNTPs,and photo-caged dNTPs. Such reagents are known and available in the art.One of skill in the art will be able to determine the optimumtemperatures as appropriate for individual reagents.

Amplification of nucleic acids may be performed using specific thermalcycle machinery or equipment, and may be performed in single reactionsor in bulk, such that any desired number of reactions may be performedsimultaneously. In some embodiments, amplification may be performedusing microfluidic or robotic devices, or may be performed using manualalteration in temperatures to achieve the desired amplification. In someembodiments, optimization may be performed to obtain the optimumreactions conditions for the particular application or materials. One ofskill in the art will understand and be able to optimize reactionconditions to obtain sufficient amplification.

In certain embodiments, detection of DNA with the methods or systems ofthe invention requires transcription of the (amplified) DNA into RNAprior to detection.

Target RNA/DNA Enrichment

In certain example embodiments, target RNA or DNA may first be enrichedprior to detection or amplification of the target RNA or DNA. In certainexample embodiments, this enrichment may be achieved by binding of thetarget nucleic acids by a CRISPR effector system.

Current target-specific enrichment protocols require single-strandednucleic acid prior to hybridization with probes. Among variousadvantages, the present embodiments can skip this step and enable directtargeting to double-stranded DNA (either partly or completelydouble-stranded). In addition, the embodiments disclosed herein areenzyme-driven targeting methods that offer faster kinetics and easierworkflow allowing for isothermal enrichment. In certain exampleembodiments enrichment may take place at temperatures as low as 20-37°C. In certain example embodiments, a set of guide RNAs to differenttarget nucleic acids are used in a single assay, allowing for detectionof multiple targets and/or multiple variants of a single target.

In certain example embodiments, a dead CRISPR effector protein may bindthe target nucleic acid in solution and then subsequently be isolatedfrom said solution. For example, the dead CRISPR effector protein boundto the target nucleic acid, may be isolated from the solution using anantibody or other molecule, such as an aptamer, that specifically bindsthe dead CRISPR effector protein.

In other example embodiments, the dead CRISPR effector protein may boundto a solid substrate. A fixed substrate may refer to any material thatis appropriate for or can be modified to be appropriate for theattachment of a polypeptide or a polynucleotide. Possible substratesinclude, but are not limited to, glass and modified functionalizedglass, plastics (including acrylics, polystyrene and copolymers ofstyrene and other materials, polypropylene, polyethylene, polybutylene,polyurethanes, Teflon™, etc.), polysaccharides, nylon or nitrocellulose,ceramics, resins, silica or silica-based materials including silicon andmodified silicon, carbon, metals, inorganic glasses, plastics, opticalfiber bundles, and a variety of other polymers. In some embodiments, thesolid support comprises a patterned surface suitable for immobilizationof molecules in an ordered pattern. In certain embodiments a patternedsurface refers to an arrangement of different regions in or on anexposed layer of a solid support. In some embodiments, the solid supportcomprises an array of wells or depressions in a surface. The compositionand geometry of the solid support can vary with its use. In someembodiments, the solids support is a planar structure such as a slide,chip, microchip and/or array. As such, the surface of the substrate canbe in the form of a planar layer. In some embodiments, the solid supportcomprises one or more surfaces of a flowcell. The term “flowcell” asused herein refers to a chamber comprising a solid surface across whichone or more fluid reagents can be flowed. Example flowcells and relatedfluidic systems and detection platforms that can be readily used in themethods of the present disclosure are described, for example, in Bentleyet al. Nature 456:53-59 (2008), WO 04/0918497, U.S. Pat. No. 7,057,026;WO 91/06678; WO 07/123744; U.S. Pat. Nos. 7,329,492; 7,211,414;7,315,019; 7,405,281, and US 2008/0108082. In some embodiments, thesolid support or its surface is non-planar, such as the inner or outersurface of a tube or vessel. In some embodiments, the solid supportcomprise microspheres or beads. “Microspheres,” “bead,” “particles,” areintended to mean within the context of a solid substrate to mean smalldiscrete particles made of various material including, but not limitedto, plastics, ceramics, glass, and plystyrene. In certain embodiments,the microspheres are magnetic microspheres or beads. Alternatively oradditionally, the beads may be porous. The bead sizes range fromnanometers, e.g. 100 nm, to millimeters, e.g. 1 mm.

A sample containing, or suspected of containing, the target nucleicacids may then be exposed to the substrate to allow binding of thetarget nucleic acids to the bound dead CRISPR effector protein.Non-target molecules may then be washed away. In certain exampleembodiments, the target nucleic acids may then be released from theCRISPR effector protein/guide RNA complex for further detection usingthe methods disclosed herein. In certain example embodiments, the targetnucleic acids may first be amplified as described herein.

In certain example embodiments, the CRISPR effector may be labeled witha binding tag. In certain example embodiments the CRISPR effector may bechemically tagged. For example, the CRISPR effector may be chemicallybiotinylated. In another example embodiment, a fusion may be created byadding additional sequence encoding a fusion to the CRISPR effector. Oneexample of such a fusion is an AviTag™, which employs a highly targetedenzymatic conjugation of a single biotin on a unique 15 amino acidpeptide tag. In certain embodiments, the CRISPR effector may be labeledwith a capture tag such as, but not limited to, GST, Myc, hemagglutinin(HA), green fluorescent protein (GFP), flag, His tag, TAP tag, and Fctag. The binding tag, whether a fusion, chemical tag, or capture tag,may be used to either pull down the CRISPR effector system once it hasbound a target nucleic acid or to fix the CRISPR effector system on thesolid substrate.

In certain example embodiments, the guide RNA may be labeled with abinding tag. In certain example embodiments, the entire guide RNA may belabeled using in vitro transcription (IVT) incorporating one or morebiotinylated nucleotides, such as, biotinylated uracil. In someembodiments, biotin can be chemically or enzymatically added to theguide RNA, such as, the addition of one or more biotin groups to the 3′end of the guide RNA. The binding tag may be used to pull down the guideRNA/target nucleic acid complex after binding has occurred, for example,by exposing the guide RNA/target nucleic acid to a streptavidin coatedsolid substrate.

Accordingly, in certain example embodiments, an engineered ornon-naturally-occurring CRISPR effector may be used for enrichmentpurposes. In an embodiment, the modification may comprise mutation ofone or more amino acid residues of the effector protein. The one or moremutations may be in one or more catalytically active domains of theeffector protein. The effector protein may have reduced or abolishednuclease activity compared with an effector protein lacking said one ormore mutations. The effector protein may not direct cleavage of the RNAstrand at the target locus of interest. In a preferred embodiment, theone or more mutations may comprise two mutations. In a preferredembodiment the one or more amino acid residues are modified in a C2c2effector protein, e.g., an engineered or non-naturally-occurringeffector protein or C2c2. In particular embodiments, the one or moremodified of mutated amino acid residues are one or more of those in C2c2corresponding to R597, H602, R1278 and H1283 (referenced to Lsh C2c2amino acids), such as mutations R597A, H602A, R1278A and H1283A, or thecorresponding amino acid residues in Lsh C2c2 orthologues.

In particular embodiments, the one or more modified of mutated aminoacid residues are one or more of those in C2c2 corresponding to K2, K39,V40, E479, L514, V518, N524, G534, K535, E580, L597, V602, D630, F676,L709, I713, R717 (HEPN), N718, H722 (HEPN), E773, P823, V828, I879,Y880, F884, Y997, L1001, F1009, L1013, Y1093, L1099, L1111, Y1114,L1203, D1222, Y1244, L1250, L1253, K1261, I1334, L1355, L1359, R1362,Y1366, E1371, R1372, D1373, R1509 (HEPN), H1514 (HEPN), Y1543, D1544,K1546, K1548, V1551, I1558, according to C2c2 consensus numbering. Incertain embodiments, the one or more modified of mutated amino acidresidues are one or more of those in C2c2 corresponding to R717 andR1509. In certain embodiments, the one or more modified of mutated aminoacid residues are one or more of those in C2c2 corresponding to K2, K39,K535, K1261, R1362, R1372, K1546 and K1548. In certain embodiments, saidmutations result in a protein having an altered or modified activity. Incertain embodiments, said mutations result in a protein having a reducedactivity, such as reduced specificity. In certain embodiments, saidmutations result in a protein having no catalytic activity (i.e. “dead”C2c2). In an embodiment, said amino acid residues correspond to Lsh C2c2amino acid residues, or the corresponding amino acid residues of a C2c2protein from a different species. Devices that can facilitate thesesteps. In some embodiments, to reduce the size of a fusion protein ofthe Cas13b effector and the one or more functional domains, theC-terminus of the Cas13b effector can be truncated while stillmaintaining its RNA binding function. For example, at least 20 aminoacids, at least 50 amino acids, at least 80 amino acids, or at least 100amino acids, or at least 150 amino acids, or at least 200 amino acids,or at least 250 amino acids, or at least 300 amino acids, or at least350 amino acids, or up to 120 amino acids, or up to 140 amino acids, orup to 160 amino acids, or up to 180 amino acids, or up to 200 aminoacids, or up to 250 amino acids, or up to 300 amino acids, or up to 350amino acids, or up to 400 amino acids, may be truncated at theC-terminus of the Cas13b effector. Specific examples of Cas13btruncations include C-terminal 4984-1090, C-terminal 41026-1090, andC-terminal 41053-1090, C-terminal 4934-1090, C-terminal 4884-1090,C-terminal 4834-1090, C-terminal 4784-1090, and C-terminal 4734-1090,wherein amino acid positions correspond to amino acid positions ofPrevotella sp. P5-125 Cas13b protein.

The above enrichment systems may also be used to deplete a sample ofcertain nucleic acids. For example, guide RNAs may be designed to bindnon-target RNAs to remove the non-target RNAs from the sample. In oneexample embodiment, the guide RNAs may be designed to bind nucleic acidsthat do carry a particular nucleic acid variation. For example, in agiven sample a higher copy number of non-variant nucleic acids may beexpected. Accordingly, the embodiments disclosed herein may be used toremove the non-variant nucleic acids from a sample, to increase theefficiency with which the detection CRISPR effector system can detectthe target variant sequences in a given sample.

Amplification and/or Enhancement of Detectable Positive Signal

In certain example embodiments, further modification may be introducedthat further amplify the detectable positive signal. For example,activated CRISPR effector protein collateral activation may be use togenerate a secondary target or additional guide sequence, or both. Inone example embodiment, the reaction solution would contain a secondarytarget that is spiked in at high concentration. The secondary target maybe distinct from the primary target (i.e. the target for which the assayis designed to detect) and in certain instances may be common across allreaction volumes. A secondary guide sequence for the secondary targetmay be protected, e.g. by a secondary structural feature such as ahairpin with a RNA loop, and unable to bind the second target or theCRISPR effector protein. Cleavage of the protecting group by anactivated CRISPR effector protein (i.e. after activation by formation ofcomplex with the primary target(s) in solution) and formation of acomplex with free CRISPR effector protein in solution and activationfrom the spiked in secondary target. In certain other exampleembodiments, a similar concept is used with a second guide sequence to asecondary target sequence. The secondary target sequence may beprotected a structural feature or protecting group on the secondarytarget. Cleavage of a protecting group off the secondary target thenallows additional CRISPR effector protein/second guidesequence/secondary target complex to form. In yet another exampleembodiment, activation of CRISPR effector protein by the primarytarget(s) may be used to cleave a protected or circularized primer,which is then released to perform an isothermal amplification reaction,such as those disclosed herein, on a template that encodes a secondaryguide sequence, secondary target sequence, or both. Subsequenttranscription of this amplified template would produce more secondaryguide sequence and/or secondary target sequence, followed by additionalCRISPR effector protein collateral activation.

Detection of Proteins

The systems, devices, and methods disclosed herein may also be adaptedfor detection of polypeptides (or other molecules) in addition todetection of nucleic acids, via incorporation of a specificallyconfigured polypeptide detection aptamer. The polypeptide detectionaptamers are distinct from the masking construct aptamers discussedabove. First, the aptamers are designed to specifically bind to one ormore target molecules. In one example embodiment, the target molecule isa target polypeptide. In another example embodiment, the target moleculeis a target chemical compound, such as a target therapeutic molecule.Methods for designing and selecting aptamers with specificity for agiven target, such as SELEX, are known in the art. In addition tospecificity to a given target the aptamers are further designed toincorporate a RNA polymerase promoter binding site. In certain exampleembodiments, the RNA polymerase promoter is a T7 promoter. Prior tobinding the apatamer binding to a target, the RNA polymerase site is notaccessible or otherwise recognizable to a RNA polymerase. However, theaptamer is configured so that upon binding of a target the structure ofthe aptamer undergoes a conformational change such that the RNApolymerase promoter is then exposed. An aptamer sequence downstream ofthe RNA polymerase promoter acts as a template for generation of atrigger RNA oligonucleotide by a RNA polymerase. Thus, the templateportion of the aptamer may further incorporate a barcode or otheridentifying sequence that identifies a given aptamer and its target.Guide RNAs as described above may then be designed to recognize thesespecific trigger oligonucleotide sequences. Binding of the guide RNAs tothe trigger oligonucleotides activates the CRISPR effector proteinswhich proceeds to deactivate the masking constructs and generate apositive detectable signal as described previously.

Accordingly, in certain example embodiments, the methods disclosedherein comprise the additional step of distributing a sample or set ofsample into a set of individual discrete volumes, each individualdiscrete volume comprising peptide detection aptamers, a CRISPR effectorprotein, one or more guide RNAs, a masking construct, and incubating thesample or set of samples under conditions sufficient to allow binding ofthe detection aptamers to the one or more target molecules, whereinbinding of the aptamer to a corresponding target results in exposure ofthe RNA polymerase promoter binding site such that synthesis of atrigger RNA is initiated by the binding of a RNA polymerase to the RNApolymerase promoter binding site.

In another example embodiment, binding of the aptamer may expose aprimer binding site upon binding of the aptamer to a target polypeptide.For example, the aptamer may expose a RPA primer binding site. Thus, theaddition or inclusion of the primer will then feed into an amplificationreaction, such as the RPA reaction outlined above.

In certain example embodiments, the aptamer may be aconformation-switching aptamer, which upon binding to the target ofinterest may change secondary structure and expose new regions ofsingle-stranded DNA. In certain example embodiments, these new-regionsof single-stranded DNA may be used as substrates for ligation, extendingthe aptamers and creating longer ssDNA molecules which can bespecifically detected using the embodiments disclosed herein. Theaptamer design could be further combined with ternary complexes fordetection of low-epitope targets, such as glucose (Yang et al. 2015:http://pubs.acs.org/doi/abs/10.1021/acs.analchem.5b01634). Exampleconformation shifting aptamers and corresponding guide RNAs (crRNAs) areshown below.

Thrombin aptamer (SEQ. I.D. No. 186) Thrombin ligation probe (SEQ. I.D.No. 187) Thrombin RPA forward 1 (SEQ. I.D. No. 188) primer Thrombin RPAforward 2 (SEQ. I.D. No. 189) primer Thrombin RPA reverse 1 (SEQ. I.D.No. 190) primer Thrombin crRNA 1 (SEQ. I.D. No. 191) Thrombin crRNA 2(SEQ. I.D. No. 192) Thrombin crRNA 3 (SEQ. I.D. No. 193) PTK7 fulllength amplicon (SEQ. I.D. No. 194) control PTK7 aptamer (SEQ. I.D. No.195) PTK7 ligation probe (SEQ. I.D. No. 196) PTK7 RPA forward 1 primer(SEQ. I.D. No. 197) PTK7 RPA reverse 1 primer (SEQ. I.D. No. 198) PTK7crRNA 1 (SEQ. I.D. No. 199) PTK7 crRNA 2 (SEQ. I.D. No. 200) PTK7 crRNA3 (SEQ. I.D. No. 201)

Devices

The systems described herein can be embodied on diagnostic devices. Anumber of substrates and configurations may be used. The devices may becapable of defining multiple individual discrete volumes within thedevice. As used herein an “individual discrete volume” refers to adiscrete space, such as a container, receptacle, or other defined volumeor space that can be defined by properties that prevent and/or inhibitmigration of target molecules, for example a volume or space defined byphysical properties such as walls, for example the walls of a well,tube, or a surface of a droplet, which may be impermeable orsemipermeable, or as defined by other means such as chemical, diffusionrate limited, electro-magnetic, or light illumination, or anycombination thereof that can contain a a sample within a defined space.Individual discrete volumes may be identified by molecular tags, such asnucleic acid barcodes. By “diffusion rate limited” (for examplediffusion defined volumes) is meant spaces that are only accessible tocertain molecules or reactions because diffusion constraints effectivelydefining a space or volume as would be the case for two parallel laminarstreams where diffusion will limit the migration of a target moleculefrom one stream to the other. By “chemical” defined volume or space ismeant spaces where only certain target molecules can exist because oftheir chemical or molecular properties, such as size, where for examplegel beads may exclude certain species from entering the beads but notothers, such as by surface charge, matrix size or other physicalproperty of the bead that can allow selection of species that may enterthe interior of the bead. By “electro-magnetically” defined volume orspace is meant spaces where the electro-magnetic properties of thetarget molecules or their supports such as charge or magnetic propertiescan be used to define certain regions in a space such as capturingmagnetic particles within a magnetic field or directly on magnets. By“optically” defined volume is meant any region of space that may bedefined by illuminating it with visible, ultraviolet, infrared, or otherwavelengths of light such that only target molecules within the definedspace or volume may be labeled. One advantage to the use of non-walled,or semipermeable discrete volumes is that some reagents, such asbuffers, chemical activators, or other agents may be passed through thediscrete volume, while other materials, such as target molecules, may bemaintained in the discrete volume or space. Typically, a discrete volumewill include a fluid medium, (for example, an aqueous solution, an oil,a buffer, and/or a media capable of supporting cell growth) suitable forlabeling of the target molecule with the indexable nucleic acididentifier under conditions that permit labeling. Exemplary discretevolumes or spaces useful in the disclosed methods include droplets (forexample, microfluidic droplets and/or emulsion droplets), hydrogel beadsor other polymer structures (for example poly-ethylene glycoldi-acrylate beads or agarose beads), tissue slides (for example, fixedformalin paraffin embedded tissue slides with particular regions,volumes, or spaces defined by chemical, optical, or physical means),microscope slides with regions defined by depositing reagents in orderedarrays or random patterns, tubes (such as, centrifuge tubes,microcentrifuge tubes, test tubes, cuvettes, conical tubes, and thelike), bottles (such as glass bottles, plastic bottles, ceramic bottles,Erlenmeyer flasks, scintillation vials and the like), wells (such aswells in a plate), plates, pipettes, or pipette tips among others. Incertain embodiments, the compartment is an aqueous droplet in awater-in-oil emulsion. In specific embodiments, any of the applications,methods, or systems described herein requiring exact or uniform volumesmay employ the use of an acoustic liquid dispenser.

In certain example embodiments, the device comprises a flexible materialsubstrate on which a number of spots may be defined. Flexible substratematerials suitable for use in diagnostics and biosensing are knownwithin the art. The flexible substrate materials may be made of plantderived fibers, such as cellulosic fibers, or may be made from flexiblepolymers such as flexible polyester films and other polymer types.Within each defined spot, reagents of the system described herein areapplied to the individual spots. Each spot may contain the same reagentsexcept for a different guide RNA or set of guide RNAs, or whereapplicable, a different detection aptamer to screen for multiple targetsat once. Thus, the systems and devices herein may be able to screensamples from multiple sources (e.g. multiple clinical samples fromdifferent individuals) for the presence of the same target, or a limitednumber of targets, or aliquots of a single sample (or multiple samplesfrom the same source) for the presence of multiple different targets inthe sample. In certain example embodiments, the elements of the systemsdescribed herein are freeze dried onto the paper or cloth substrate.Example flexible material based substrates that may be used in certainexample devices are disclosed in Pardee et al. Cell. 2016,165(5):1255-66 and Pardee et al. Cell. 2014, 159(4):950-54. Suitableflexible material-based substrates for use with biological fluids,including blood are disclosed in International Patent ApplicationPublication No. WO/2013/071301 entitled “Paper based diagnostic test” toShevkoplyas et al. U.S. Patent Application Publication No. 2011/0111517entitled “Paper-based microfluidic systems” to Siegel et al. and Shafieeet al. “Paper and Flexible Substrates as Materials for BiosensingPlatforms to Detect Multiple Biotargets” Scientific Reports 5:8719(2015). Further flexible based materials, including those suitable foruse in wearable diagnostic devices are disclosed in Wang et al.“Flexible Substrate-Based Devices for Point-of-Care Diagnostics” Cell34(11):909-21 (2016). Further flexible based materials may includenitrocellulose, polycarbonate, methylethyl cellulose, polyvinylidenefluoride (PVDF), polystyrene, or glass (see e.g., US20120238008). Incertain embodiments, discrete volumes are separated by a hydrophobicsurface, such as but not limited to wax, photoresist, or solid ink.

In some embodiments, a dosimeter or badge may be provided that serves asa sensor or indicator such that the wearer is notified of exposure tocertain microbes or other agents. For example, the systems describedherein may be used to detect a particular pathogen. Likewise, aptamerbased embodiments disclosed above may be used to detect both polypeptideas well as other agents, such as chemical agents, to which a specificaptamer may bind. Such a device may be useful for surveillance ofsoldiers or other military personnel, as well as clinicians,researchers, hospital staff, and the like, in order to provideinformation relating to exposure to potentially dangerous agents asquickly as possible, for example for biological or chemical warfareagent detection. In other embodiments, such a surveillance badge may beused for preventing exposure to dangerous microbes or pathogens inimmunocompromised patients, burn patients, patients undergoingchemotherapy, children, or elderly individuals.

Samples sources that may be analyzed using the systems and devicesdescribed herein include biological samples of a subject orenvironmental samples. Environmental samples may include surfaces orfluids. The biological samples may include, but are not limited to,saliva, blood, plasma, sera, stool, urine, sputum, mucous, lymph,synovial fluid, spinal fluid, cerebrospinal fluid, a swab from skin or amucosal membrane, or combination thereof. In an example embodiment, theenvironmental sample is taken from a solid surface, such as a surfaceused in the preparation of food or other sensitive compositions andmaterials.

In other example embodiments, the elements of the systems describedherein may be place on a single use substrate, such as swab or cloththat is used to swab a surface or sample fluid. For example, the systemcould be used to test for the presence of a pathogen on a food byswabbing the surface of a food product, such as a fruit or vegetable.Similarly, the single use substrate may be used to swab other surfacesfor detection of certain microbes or agents, such as for use in securityscreening. Single use substrates may also have applications inforensics, where the CRISPR systems are designed to detect, for exampleidentifying DNA SNPs that may be used to identify a suspect, or certaintissue or cell markers to determine the type of biological matterpresent in a sample. Likewise, the single use substrate could be used tocollect a sample from a patient—such as a saliva sample from themouth—or a swab of the skin. In other embodiments, a sample or swab maybe taken of a meat product on order to detect the presence of absence ofcontaminants on or within the meat product.

Near-real-time microbial diagnostics are needed for food, clinical,industrial, and other environmental settings (see e.g., Lu T K, BowersJ, and Koeris M S., Trends Biotechnol. 2013 June; 31(6):325-7). Incertain embodiments, the present invention is used for rapid detectionof foodborne pathogens using guide RNAs specific to a pathogen (e.g.,Campylobacter jejuni, Clostridium perfringens, Salmonella spp.,Escherichia coli, Bacillus cereus, Listeria monocytogenes, Shigellaspp., Staphylococcus aureus, Staphylococcal enteritis, Streptococcus,Vibrio cholerae, Vibrio parahaemolyticus, Vibrio vulnificus, Yersiniaenterocolitica and Yersinia pseudotuberculosis, Brucella spp.,Corynebacterium ulcerans, Coxiella burnetii, or Plesiomonasshigelloides).

In certain embodiments, the device is or comprises a flow strip. Forinstance, a lateral flow strip allows for RNAse (e.g. C2c2) detection bycolor. The RNA reporter is modified to have a first molecule (such asfor instance FITC) attached to the 5′ end and a second molecule (such asfor instance biotin) attached to the 3′ end (or vice versa). The lateralflow strip is designed to have two capture lines with anti-firstmolecule (e.g. anti-FITC) antibodies hybridized at the first line andanti-second molecule (e.g. anti-biotin) antibodies at the seconddownstream line. As the reaction flows down the strip, uncleavedreporter will bind to anti-first molecule antibodies at the firstcapture line, while cleaved reporters will liberate the second moleculeand allow second molecule binding at the second capture line. Secondmolecule sandwich antibodies, for instance conjugated to nanoparticles,such as gold nanoparticles, will bind any second molecule at the firstor second line and result in a strong readout/signal (e.g. color). Asmore reporter is cleaved, more signal will accumulate at the secondcapture line and less signal will appear at the first line. In certainaspects, the invention relates to the use of a follow strip as describedherein for detecting nucleic acids or polypeptides. In certain aspects,the invention relates to a method for detecting nucleic acids orpolypeptides with a flow strip as defined herein, e.g. (lateral) flowtests or (lateral) flow immunochromatographic assays.

In certain example embodiments, the device is a microfluidic device thatgenerates and/or merges different droplets (i.e. individual discretevolumes). For example, a first set of droplets may be formed containingsamples to be screened and a second set of droplets formed containingthe elements of the systems described herein. The first and second setof droplets are then merged and then diagnostic methods as describedherein are carried out on the merged droplet set. Microfluidic devicesdisclosed herein may be silicone-based chips and may be fabricated usinga variety of techniques, including, but not limited to, hot embossing,molding of elastomers, injection molding, LIGA, soft lithography,silicon fabrication and related thin film processing techniques.Suitable materials for fabricating the microfluidic devices include, butare not limited to, cyclic olefin copolymer (COC), polycarbonate,poly(dimethylsiloxane) (PDMS), and poly(methylacrylate) (PMMA). In oneembodiment, soft lithography in PDMS may be used to prepare themicrofluidic devices. For example, a mold may be made usingphotolithography which defines the location of flow channels, valves,and filters within a substrate. The substrate material is poured into amold and allowed to set to create a stamp. The stamp is then sealed to asolid support, such as but not limited to, glass. Due to the hydrophobicnature of some polymers, such as PDMS, which absorbs some proteins andmay inhibit certain biological processes, a passivating agent may benecessary (Schoffner et al. Nucleic Acids Research, 1996, 24:375-379).Suitable passivating agents are known in the art and include, but arenot limited to, silanes, parylene, n-Dodecyl-b-D-matoside (DDM),pluronic, Tween-20, other similar surfactants, polyethylene glycol(PEG), albumin, collagen, and other similar proteins and peptides.

In certain example embodiments, the system and/or device may be adaptedfor conversion to a flow-cytometry readout in or allow to all ofsensitive and quantitative measurements of millions of cells in a singleexperiment and improve upon existing flow-based methods, such as thePrimeFlow assay. In certain example embodiments, cells may be cast indroplets containing unpolymerized gel monomer, which can then be castinto single-cell droplets suitable for analysis by flow cytometry. Adetection construct comprising a fluorescent detectable label may becast into the droplet comprising unpolymerized gel monomer. Uponpolymerization of the gel monomer to form a bead within a droplet.Because gel polymerization is through free-radical formation, thefluorescent reporter becomes covalently bound to the gel. The detectionconstruct may be further modified to comprise a linker, such as anamine. A quencher may be added post-gel formation and will bind via thelinker to the reporter construct. Thus, the quencher is not bound to thegel and is free to diffuse away when the reporter is cleaved by theCRISPR effector protein. Amplification of signal in droplet may beachieved by coupling the detection construct to a hybridization chainreaction (HCR initiator) amplification. DNA/RNA hybrid hairpins may beincorporated into the gel which may comprise a hairpin loop that has aRNase sensitive domain. By protecting a strand displacement toeholdwithin a hairpin loop that has a RNase sensitive domain, HCR initiatorsmay be selectively deprotected following cleavage of the hairpin loop bythe CRISPR effector protein. Following deprotection of HCR initiatorsvia toehold mediated strand displacement, fluorescent HCR monomers maybe washed into the gel to enable signal amplification where theinitiators are deprotected.

An example of microfluidic device that may be used in the context of theinvention is described in Hour et al. “Direct Detection anddrug-resistance profiling of bacteremias using inertial microfluidics”Lap Chip. 15(10):2297-2307 (2016).

In systems described herein, may further be incorporated into wearablemedical devices that assess biological samples, such as biologicalfluids, of a subject outside the clinic setting and report the outcomeof the assay remotely to a central server accessible by a medical careprofessional. The device may include the ability to self-sample blood,such as the devices disclosed in U.S. Patent Application Publication No.2015/0342509 entitled “Needle-free Blood Draw to Peeters et al., U.S.Patent Application Publication No. 2015/0065821 entitled “NanoparticlePhoresis” to Andrew Conrad.

In certain example embodiments, the device may comprise individualwells, such as microplate wells. The size of the microplate wells may bethe size of standard 6, 24, 96, 384, 1536, 3456, or 9600 sized wells. Incertain example embodiments, the elements of the systems describedherein may be freeze dried and applied to the surface of the well priorto distribution and use.

The devices disclosed herein may further comprise inlet and outletports, or openings, which in turn may be connected to valves, tubes,channels, chambers, and syringes and/or pumps for the introduction andextraction of fluids into and from the device. The devices may beconnected to fluid flow actuators that allow directional movement offluids within the microfluidic device. Example actuators include, butare not limited to, syringe pumps, mechanically actuated recirculatingpumps, electroosmotic pumps, bulbs, bellows, diaphragms, or bubblesintended to force movement of fluids. In certain example embodiments,the devices are connected to controllers with programmable valves thatwork together to move fluids through the device. In certain exampleembodiments, the devices are connected to the controllers discussed infurther detail below. The devices may be connected to flow actuators,controllers, and sample loading devices by tubing that terminates inmetal pins for insertion into inlet ports on the device.

As shown herein the elements of the system are stable when freeze dried,therefore embodiments that do not require a supporting device are alsocontemplated, i.e. the system may be applied to any surface or fluidthat will support the reactions disclosed herein and allow for detectionof a positive detectable signal from that surface or solution. Inaddition to freeze-drying, the systems may also be stably stored andutilized in a pelletized form. Polymers useful in forming suitablepelletized forms are known in the art.

In certain embodiments, the CRISPR effector protein is bound to eachdiscrete volume in the device. Each discrete volume may comprise adifferent guide RNA specific for a different target molecule. In certainembodiments, a sample is exposed to a solid substrate comprising morethan one discrete volume each comprising a guide RNA specific for atarget molecule. Not being bound by a theory, each guide RNA willcapture its target molecule from the sample and the sample does not needto be divided into separate assays. Thus, a valuable sample may bepreserved. The effector protein may be a fusion protein comprising anaffinity tag. Affinity tags are well known in the art (e.g., HA tag, Myctag, Flag tag, His tag, biotin). The effector protein may be linked to abiotin molecule and the discrete volumes may comprise streptavidin. Inother embodiments, the CRISPR effector protein is bound by an antibodyspecific for the effector protein. Methods of binding a CRISPR enzymehas been described previously (see, e.g., US20140356867A1).

The devices disclosed herein may also include elements of point of care(POC) devices known in the art for analyzing samples by other methods.See, for example St John and Price, “Existing and Emerging Technologiesfor Point-of-Care Testing” (Clin Biochem Rev. 2014 August; 35(3):155-167).

The present invention may be used with a wireless lab-on-chip (LOC)diagnostic sensor system (see e.g., U.S. Pat. No. 9,470,699 “Diagnosticradio frequency identification sensors and applications thereof”). Incertain embodiments, the present invention is performed in a LOCcontrolled by a wireless device (e.g., a cell phone, a personal digitalassistant (PDA), a tablet) and results are reported to said device.

Radio frequency identification (RFID) tag systems include an RFID tagthat transmits data for reception by an RFID reader (also referred to asan interrogator). In a typical RFID system, individual objects (e.g.,store merchandise) are equipped with a relatively small tag thatcontains a transponder. The transponder has a memory chip that is givena unique electronic product code. The RFID reader emits a signalactivating the transponder within the tag through the use of acommunication protocol. Accordingly, the RFID reader is capable ofreading and writing data to the tag. Additionally, the RFID tag readerprocesses the data according to the RFID tag system application.Currently, there are passive and active type RFID tags. The passive typeRFID tag does not contain an internal power source, but is powered byradio frequency signals received from the RFID reader. Alternatively,the active type RFID tag contains an internal power source that enablesthe active type RFID tag to possess greater transmission ranges andmemory capacity. The use of a passive versus an active tag is dependentupon the particular application.

Lab-on-the chip technology is well described in the scientificliterature and consists of multiple microfluidic channels, input orchemical wells. Reactions in wells can be measured using radio frequencyidentification (RFID) tag technology since conductive leads from RFIDelectronic chip can be linked directly to each of the test wells. Anantenna can be printed or mounted in another layer of the electronicchip or directly on the back of the device. Furthermore, the leads, theantenna and the electronic chip can be embedded into the LOC chip,thereby preventing shorting of the electrodes or electronics. Since LOCallows complex sample separation and analyses, this technology allowsLOC tests to be done independently of a complex or expensive reader.Rather a simple wireless device such as a cell phone or a PDA can beused. In one embodiment, the wireless device also controls theseparation and control of the microfluidics channels for more complexLOC analyses. In one embodiment, a LED and other electronic measuring orsensing devices are included in the LOC-RFID chip. Not being bound by atheory, this technology is disposable and allows complex tests thatrequire separation and mixing to be performed outside of a laboratory.

In preferred embodiments, the LOC may be a microfluidic device. The LOCmay be a passive chip, wherein the chip is powered and controlledthrough a wireless device. In certain embodiments, the LOC includes amicrofluidic channel for holding reagents and a channel for introducinga sample. In certain embodiments, a signal from the wireless devicedelivers power to the LOC and activates mixing of the sample and assayreagents. Specifically, in the case of the present invention, the systemmay include a masking agent, CRISPR effector protein, and guide RNAsspecific for a target molecule. Upon activation of the LOC, themicrofluidic device may mix the sample and assay reagents. Upon mixing,a sensor detects a signal and transmits the results to the wirelessdevice. In certain embodiments, the unmasking agent is a conductive RNAmolecule. The conductive RNA molecule may be attached to the conductivematerial. Conductive molecules can be conductive nanoparticles,conductive proteins, metal particles that are attached to the protein orlatex or other beads that are conductive. In certain embodiments, if DNAor RNA is used then the conductive molecules can be attached directly tothe matching DNA or RNA strands. The release of the conductive moleculesmay be detected across a sensor. The assay may be a one step process.

Since the electrical conductivity of the surface area can be measuredprecisely quantitative results are possible on the disposable wirelessRFID electro-assays. Furthermore, the test area can be very smallallowing for more tests to be done in a given area and thereforeresulting in cost savings. In certain embodiments, separate sensors eachassociated with a different CRISPR effector protein and guide RNAimmobilized to a sensor are used to detect multiple target molecules.Not being bound by a theory, activation of different sensors may bedistinguished by the wireless device.

In addition to the conductive methods described herein, other methodsmay be used that rely on RFID or Bluetooth as the basic low costcommunication and power platform for a disposable RFID assay. Forexample, optical means may be used to assess the presence and level of agiven target molecule. In certain embodiments, an optical sensor detectsunmasking of a fluorescent masking agent.

In certain embodiments, the device of the present invention may includehandheld portable devices for diagnostic reading of an assay (see e.g.,Vashist et al., Commercial Smartphone-Based Devices and SmartApplications for Personalized Healthcare Monitoring and Management,Diagnostics 2014, 4(3), 104-128; mReader from Mobile Assay; and HolomicRapid Diagnostic Test Reader).

As noted herein, certain embodiments allow detection via colorimetricchange which has certain attendant benefits when embodiments areutilized in POC situations and or in resource poor environments whereaccess to more complex detection equipment to readout the signal may belimited. However, portable embodiments disclosed herein may also becoupled with hand-held spectrophotometers that enable detection ofsignals outside the visible range. An example of a hand-heldspectrophotometer device that may be used in combination with thepresent invention is described in Das et al. “Ultra-portable, wirelesssmartphone spectrophotometer for rapid, non-destructive testing of fruitripeness.” Nature Scientific Reports. 2016, 6:32504, DOI:10.1038/srep32504. Finally, in certain embodiments utilizing quantumdot-based masking constructs, use of a hand held UV light, or othersuitable device, may be successfully used to detect a signal owing tothe near complete quantum yield provided by quantum dots.

Example Methods and Assays

The low cost and adaptability of the assay platform lends itself to anumber of applications including (i) general RNA/DNA/proteinquantitation, (ii) rapid, multiplexed RNA/DNA and protein expressiondetection, and (iii) sensitive detection of target nucleic acids,peptides, and proteins in both clinical and environmental samples.Additionally, the systems disclosed herein may be adapted for detectionof transcripts within biological settings, such as cells. Given thehighly specific nature of the CRISPR effectors described herein, it maypossible to track allelic specific expression of transcripts ordisease-associated mutations in live cells.

In certain example embodiments, a single guide sequences specific to asingle target is placed in separate volumes. Each volume may thenreceive a different sample or aliquot of the same sample. In certainexample embodiments, multiple guide sequences each to separate targetmay be placed in a single well such that multiple targets may bescreened in a different well. In order to detect multiple guide RNAs ina single volume, in certain example embodiments, multiple effectorproteins with different specificities may be used. For example,different orthologs with different sequence specificities may be used.For example, one orthologue may preferentially cut A, while otherspreferentially cut C, G, U/T. Accordingly, masking constructs completelycomprising, or comprised of a substantial portion, of a singlenucleotide may be generated, each with a different fluorophore that canbe detected at differing wavelengths. In this way up to four differenttargets may be screened in a single individual discrete volume. Incertain example embodiments, different orthologues from a same class ofCRISPR effector protein may be used, such as two Cas13a orthologues, twoCas13b orthologues, or two Cas13c orthologues. The nucleotidepreferences of various Cas13 proteins is shown in FIGS. 67A and 67B. Incertain other example embodiments, different orthologues with differentnucleotide editing preferences may be used such as a Cas13a and Cas13borthologs, or a Cas13a and a Cas13c orthologs, or a Cas13b orthologs anda Cas13c orthologs etc. In certain example embodiments, a Cas13 proteinwith a polyU preference and a Cas13 protein with a polyA preference areused. In certain example embodiments, the Cas13 protein with a polyUpreference is a Prevotella intermedia Cas13b. and the Cas13 protein witha polyA preference is a Prevotella sp. MA2106 Cas13b protein(PsmCas13b). In certain example embodiments, the Cas13 protein with apolyU preference is a Leptotrichia wadei Cas13a (LwaCas13a) protein andthe Cas13 protein with a poly A preference is a Prevotella sp. MA2106Cas13b protein. In certain example embodiments, the Cas13 protein with apolyU preference is Capnocytophaga canimorsus Cas13b protein(CcaCas13b).

In addition to single base editing preferences. Additional detectionconstructs can be designed based on other motif cutting preferences ofCas 13 ortholgs. For example, Cas13 orthologs may preferentially cut adinucleotide sequence, a trinucleotide sequence or more complex motifscomprising 4, 5, 6, 7, 8, 9, or 10 nucleotide motifs. Thus the upperbound for multiplex assays using the embodiments disclosed herein isprimarily limited by the number of distinguishable detectable labels.Example methods for identifying such motifs are further disclosed in theWorking Examples below.

As demonstrated herein, the CRISPR effector systems are capable ofdetecting down to attomolar concentrations of target molecules. See e.g.FIGS. 13, 14, 19, 22 and the Working Examples described below. Due tothe sensitivity of said systems, a number of applications that requirerapid and sensitive detection may benefit from the embodiments disclosedherein, and are contemplated to be within the scope of the invention.Example assays and applications are described in further detail below.

Microbial Applications

In certain example embodiments, the systems, devices, and methods,disclosed herein are directed to detecting the presence of one or moremicrobial agents in a sample, such as a biological sample obtained froma subject. In certain example embodiments, the microbe may be abacterium, a fungus, a yeast, a protozoa, a parasite, or a virus.Accordingly, the methods disclosed herein can be adapted for use inother methods (or in combination) with other methods that require quickidentification of microbe species, monitoring the presence of microbialproteins (antigens), antibodies, antibody genes, detection of certainphenotypes (e.g. bacterial resistance), monitoring of diseaseprogression and/or outbreak, and antibiotic screening. Because of therapid and sensitive diagnostic capabilities of the embodiments disclosedhere, detection of microbe species type, down to a single nucleotidedifference, and the ability to be deployed as a POC device, theembodiments disclosed herein may be used guide therapeutic regimens,such as selection of the appropriate antibiotic or antiviral. Theembodiments disclosed herein may also be used to screen environmentalsamples (air, water, surfaces, food etc.) for the presence of microbialcontamination.

Disclosed is a method to identify microbial species, such as bacterial,viral, fungal, yeast, or parasitic species, or the like. Particularembodiments disclosed herein describe methods and systems that willidentify and distinguish microbial species within a single sample, oracross multiple samples, allowing for recognition of many differentmicrobes. The present methods allow the detection of pathogens anddistinguishing between two or more species of one or more organisms,e.g., bacteria, viruses, yeast, protozoa, and fungi or a combinationthereof, in a biological or environmental sample, by detecting thepresence of a target nucleic acid sequence in the sample. A positivesignal obtained from the sample indicates the presence of the microbe.Multiple microbes can be identified simultaneously using the methods andsystems of the invention, by employing the use of more than one effectorprotein, wherein each effector protein targets a specific microbialtarget sequence. In this way, a multi-level analysis can be performedfor a particular subject in which any number of microbes can be detectedat once. In some embodiments, simultaneous detection of multiplemicrobes may be performed using a set of probes that can identify one ormore microbial species.

Multiplex analysis of samples enables large-scale detection of samples,reducing the time and cost of analyses. However, multiplex analyses areoften limited by the availability of a biological sample. In accordancewith the invention, however, alternatives to multiplex analysis may beperformed such that multiple effector proteins can be added to a singlesample and each masking construct may be combined with a separatequencher dye. In this case, positive signals may be obtained from eachquencher dye separately for multiple detection in a single sample.

Disclosed herein are methods for distinguishing between two or morespecies of one or more organisms in a sample. The methods are alsoamenable to detecting one or more species of one or more organisms in asample.

Microbe Detection

In some embodiments, a method for detecting microbes in samples isprovided comprising distributing a sample or set of samples into one ormore individual discrete volumes, the individual discrete volumescomprising a CRISPR system as described herein; incubating the sample orset of samples under conditions sufficient to allow binding of the oneor more guide RNAs to one or more microbe-specific targets; activatingthe CRISPR effector protein via binding of the one or more guide RNAs tothe one or more target molecules, wherein activating the CRISPR effectorprotein results in modification of the RNA-based masking construct suchthat a detectable positive signal is generated; and detecting thedetectable positive signal, wherein detection of the detectable positivesignal indicates a presence of one or more target molecules in thesample. The one or more target molecules may be mRNA, gDNA (coding ornon-coding), trRNA, or rRNA comprising a target nucleotide tide sequencethat may be used to distinguish two or more microbial species/strainsfrom one another. The guide RNAs may be designed to detect targetsequences. The embodiments disclosed herein may also utilize certainsteps to improve hybridization between guide RNA and target RNAsequences. Methods for enhancing ribonucleic acid hybridization aredisclosed in WO 2015/085194, entitled “Enhanced Methods of RibonucleicAcid Hybridization” which is incorporated herein by reference. Themicrobe-specific target may be RNA or DNA or a protein. If DNA methodmay further comprise the use of DNA primers that introduce a RNApolymerase promoter as described herein. If the target is a protein thanthe method will utilized aptamers and steps specific to proteindetection described herein.

Detection of Single Nucleotide Variants

In some embodiments, one or more identified target sequences may bedetected using guide RNAs that are specific for and bind to the targetsequence as described herein. The systems and methods of the presentinvention can distinguish even between single nucleotide polymorphismspresent among different microbial species and therefore, use of multipleguide RNAs in accordance with the invention may further expand on orimprove the number of target sequences that may be used to distinguishbetween species. For example, in some embodiments, the one or more guideRNAs may distinguish between microbes at the species, genus, family,order, class, phylum, kingdom, or phenotype, or a combination thereof.

Detection Based on rRNA Sequences

In certain example embodiments, the devices, systems, and methodsdisclosed herein may be used to distinguish multiple microbial speciesin a sample. In certain example embodiments, identification may be basedon ribosomal RNA sequences, including the 16S, 23S, and 5S subunits.Methods for identifying relevant rRNA sequences are disclosed in U.S.Patent Application Publication No. 2017/0029872. In certain exampleembodiments, a set of guide RNA may designed to distinguish each speciesby a variable region that is unique to each species or strain. GuideRNAs may also be designed to target RNA genes that distinguish microbesat the genus, family, order, class, phylum, kingdom levels, or acombination thereof. In certain example embodiments where amplificationis used, a set of amplification primers may be designed to flankingconstant regions of the ribosomal RNA sequence and a guide RNA designedto distinguish each species by a variable internal region. In certainexample embodiments, the primers and guide RNAs may be designed toconserved and variable regions in the 16S subunit respectfully. Othergenes or genomic regions that uniquely variable across species or asubset of species such as the RecA gene family, RNA polymerase βsubunit, may be used as well. Other suitable phylogenetic markers, andmethods for identifying the same, are discussed for example in Wu et al.arXiv:1307.8690 [q-bio.GN].

In certain example embodiments, a method or diagnostic is designed toscreen microbes across multiple phylogenetic and/or phenotypic levels atthe same time. For example, the method or diagnostic may comprise theuse of multiple CRISPR systems with different guide RNAs. A first set ofguide RNAs may distinguish, for example, between mycobacteria, grampositive, and gram negative bacteria. These general classes can be evenfurther subdivided. For example, guide RNAs could be designed and usedin the method or diagnostic that distinguish enteric and non-entericwithin gram negative bacteria. A second set of guide RNA can be designedto distinguish microbes at the genus or species level. Thus a matrix maybe produced identifying all mycobacteria, gram positive, gram negative(further divided into enteric and non-enteric) with each genus ofspecies of bacteria identified in a given sample that fall within one ofthose classes. The foregoing is for example purposes only. Other meansfor classifying other microbe types are also contemplated and wouldfollow the general structure described above.

Screening for Drug Resistance

In certain example embodiments, the devices, systems and methodsdisclosed herein may be used to screen for microbial genes of interest,for example antibiotic and/or antiviral resistance genes. Guide RNAs maybe designed to distinguish between known genes of interest. Samples,including clinical samples, may then be screened using the embodimentsdisclosed herein for detection of such genes. The ability to screen fordrug resistance at POC would have tremendous benefit in selecting anappropriate treatment regime. In certain example embodiments, theantibiotic resistance genes are carbapenemases including KPC, NDM1,CTX-M15, OXA-48. Other antibiotic resistance genes are known and may befound for example in the Comprehensive Antibiotic Resistance Database(Jia et al. “CARD 2017: expansion and model-centric curation of theComprehensive Antibiotic Resistance Database.” Nucleic Acids Research,45, D566-573).

Ribavirin is an effective antiviral that hits a number of RNA viruses.Several clinically important viruses have evolved ribavirin resistanceincluding Foot and Mouth Disease Virus doi:10.1128/JVI.03594-13; poliovirus (Pfeifer and Kirkegaard. PNAS, 100(12):7289-7294, 2003); andhepatitis C virus (Pfeiffer and Kirkegaard, J. Virol. 79(4):2346-2355,2005). A number of other persistent RNA viruses, such as hepatitis andHIV, have evolved resistance to existing antiviral drugs: hepatitis Bvirus (lamivudine, tenofovir, entecavir) doi:10/1002/hep22900; hepatitisC virus (telaprevir, BILN2061, ITMN-191, SCh6, boceprevir, AG-021541,ACH-806) doi:10.1002/hep.22549; and HIV (many drug resistance mutations)hivb. standford.edu. The embodiments disclosed herein may be used todetect such variants among others.

Aside from drug resistance, there are a number of clinically relevantmutations that could be detected with the embodiments disclosed herein,such as persistent versus acute infection in LCMV(doi:10.1073/pnas.1019304108), and increased infectivity of Ebola (Diehlet al. Cell. 2016, 167(4):1088-1098.

As described herein elsewhere, closely related microbial species (e.g.having only a single nucleotide difference in a given target sequence)may be distinguished by introduction of a synthetic mismatch in thegRNA.

Set Cover Approaches

In particular embodiments, a set of guide RNAs is designed that canidentify, for example, all microbial species within a defined set ofmicrobes. In certain example embodiments, the methods for generatingguide RNAs as described herein may be compared to methods disclosed inWO 2017/040316, incorporated herein by reference. As described in WO2017040316, a set cover solution may identify the minimal number oftarget sequences probes or guide RNAs needed to cover an entire targetsequence or set of target sequences, e.g. a set of genomic sequences.Set cover approaches have been used previously to identify primersand/or microarray probes, typically in the 20 to 50 base pair range.See, e.g. Pearson et al.,cs.virginia.edu/˜robins/papers/primers_dam11_final.pdf., Jabado et al.Nucleic Acids Res. 2006 34(22):6605-11, Jabado et al. Nucleic Acids Res.2008, 36(1):e3 doi10.1093/nar/gkm1106, Duitama et al. Nucleic Acids Res.2009, 37(8):2483-2492, Phillippy et al. BMC Bioinformatics. 2009, 10:293doi:10.1186/1471-2105-10-293. However, such approaches generallyinvolved treating each primer/probe as k-mers and searching for exactmatches or allowing for inexact matches using suffix arrays. Inaddition, the methods generally take a binary approach to detectinghybridization by selecting primers or probes such that each inputsequence only needs to be bound by one primer or probe and the positionof this binding along the sequence is irrelevant. Alternative methodsmay divide a target genome into pre-defined windows and effectivelytreat each window as a separate input sequence under the binaryapproach—i.e. they determine whether a given probe or guide RNA bindswithin each window and require that all of the windows be bound by thestate of some probe or guide RNA. Effectively, these approaches treateach element of the “universe” in the set cover problem as being eitheran entire input sequence or a pre-defined window of an input sequence,and each element is considered “covered” if the start of a probe orguide RNA binds within the element. These approaches limit the fluidityto which different probe or guide RNA designs are allowed to cover agiven target sequence.

In contrast, the embodiments disclosed herein are directed to detectinglonger probe or guide RNA lengths, for example, in the range of 70 bp to200 bp that are suitable for hybrid selection sequencing. In addition,the methods disclosed WO 2017/040316 herein may be applied to take apan-target sequence approach capable of defining a probe or guide RNAsets that can identify and facilitate the detection sequencing of allspecies and/or strains sequences in a large and/or variable targetsequence set. For example, the methods disclosed herein may be used toidentify all variants of a given virus, or multiple different viruses ina single assay. Further, the method disclosed herein treat each elementof the “universe” in the set cover problem as being a nucleotide of atarget sequence, and each element is considered “covered” as long as aprobe or guide RNA binds to some segment of a target genome thatincludes the element. These type of set cover methods may be usedinstead of the binary approach of previous methods, the methodsdisclosed in herein better model how a probe or guide RNA may hybridizeto a target sequence. Rather than only asking if a given guide RNAsequence does or does not bind to a given window, such approaches may beused to detect a hybridization pattern—i.e. where a given probe or guideRNA binds to a target sequence or target sequences—and then determinesfrom those hybridization patterns the minimum number of probes or guideRNAs needed to cover the set of target sequences to a degree sufficientto enable both enrichment from a sample and sequencing of any and alltarget sequences. These hybridization patterns may be determined bydefining certain parameters that minimize a loss function, therebyenabling identification of minimal probe or guide RNA sets in a way thatallows parameters to vary for each species, e.g. to reflect thediversity of each species, as well as in a computationally efficientmanner that cannot be achieved using a straightforward application of aset cover solution, such as those previously applied in the probe orguide RNA design context.

The ability to detect multiple transcript abundances may allow for thegeneration of unique microbial signatures indicative of a particularphenotype. Various machine learning techniques may be used to derive thegene signatures. Accordingly, the guide RNAs of the CRISPR systems maybe used to identify and/or quantitate relative levels of biomarkersdefined by the gene signature in order to detect certain phenotypes. Incertain example embodiments, the gene signature indicates susceptibilityto an antibiotic, resistance to an antibiotic, or a combination thereof.

In one aspect of the invention, a method comprises detecting one or morepathogens. In this manner, differentiation between infection of asubject by individual microbes may be obtained. In some embodiments,such differentiation may enable detection or diagnosis by a clinician ofspecific diseases, for example, different variants of a disease.Preferably the pathogen sequence is a genome of the pathogen or afragment thereof. The method further may comprise determining theevolution of the pathogen. Determining the evolution of the pathogen maycomprise identification of pathogen mutations, e.g. nucleotide deletion,nucleotide insertion, nucleotide substitution. Amongst the latter, thereare non-synonymous, synonymous, and noncoding substitutions. Mutationsare more frequently non-synonymous during an outbreak. The method mayfurther comprise determining the substitution rate between two pathogensequences analyzed as described above. Whether the mutations aredeleterious or even adaptive would require functional analysis, however,the rate of non-synonymous mutations suggests that continued progressionof this epidemic could afford an opportunity for pathogen adaptation,underscoring the need for rapid containment. Thus, the method mayfurther comprise assessing the risk of viral adaptation, wherein thenumber non-synonymous mutations is determined. (Gire, et al., Science345, 1369, 2014).

Monitoring Microbe Outbreaks

In some embodiments, a CRISPR system or methods of use thereof asdescribed herein may be used to determine the evolution of a pathogenoutbreak. The method may comprise detecting one or more target sequencesfrom a plurality of samples from one or more subjects, wherein thetarget sequence is a sequence from a microbe causing the outbreaks. Sucha method may further comprise determining a pattern of pathogentransmission, or a mechanism involved in a disease outbreak caused by apathogen.

The pattern of pathogen transmission may comprise continued newtransmissions from the natural reservoir of the pathogen orsubject-to-subject transmissions (e.g. human-to-human transmission)following a single transmission from the natural reservoir or a mixtureof both. In one embodiment, the pathogen transmission may be bacterialor viral transmission, in such case, the target sequence is preferably amicrobial genome or fragments thereof. In one embodiment, the pattern ofthe pathogen transmission is the early pattern of the pathogentransmission, i.e. at the beginning of the pathogen outbreak.Determining the pattern of the pathogen transmission at the beginning ofthe outbreak increases likelihood of stopping the outbreak at theearliest possible time thereby reducing the possibility of local andinternational dissemination.

Determining the pattern of the pathogen transmission may comprisedetecting a pathogen sequence according to the methods described herein.Determining the pattern of the pathogen transmission may furthercomprise detecting shared intra-host variations of the pathogen sequencebetween the subjects and determining whether the shared intra-hostvariations show temporal patterns. Patterns in observed intrahost andinterhost variation provide important insight about transmission andepidemiology (Gire, et al., 2014).

Detection of shared intra-host variations between the subjects that showtemporal patterns is an indication of transmission links between subject(in particular between humans) because it can be explained by subjectinfection from multiple sources (superinfection), sample contaminationrecurring mutations (with or without balancing selection to reinforcemutations), or co-transmission of slightly divergent viruses that aroseby mutation earlier in the transmission chain (Park, et al., Cell161(7):1516-1526, 2015). Detection of shared intra-host variationsbetween subjects may comprise detection of intra-host variants locatedat common single nucleotide polymorphism (SNP) positions. Positivedetection of intra-host variants located at common (SNP) positions isindicative of superinfection and contamination as primary explanationsfor the intra-host variants. Superinfection and contamination can beparted on the basis of SNP frequency appearing as inter-host variants(Park, et al., 2015). Otherwise superinfection and contamination can beruled out. In this latter case, detection of shared intra-hostvariations between subjects may further comprise assessing thefrequencies of synonymous and nonsynonymous variants and comparing thefrequency of synonymous and nonsynonymous variants to one another. Anonsynonymous mutation is a mutation that alters the amino acid of theprotein, likely resulting in a biological change in the microbe that issubject to natural selection. Synonymous substitution does not alter anamino acid sequence. Equal frequency of synonymous and nonsynonymousvariants is indicative of the intra-host variants evolving neutrally. Iffrequencies of synonymous and nonsynonymous variants are divergent, theintra-host variants are likely to be maintained by balancing selection.If frequencies of synonymous and nonsynonymous variants are low, this isindicative of recurrent mutation. If frequencies of synonymous andnonsynonymous variants are high, this is indicative of co-transmission(Park, et al., 2015).

Like Ebola virus, Lassa virus (LASV) can cause hemorrhagic fever withhigh case fatality rates. Andersen et al. generated a genomic catalog ofalmost 200 LASV sequences from clinical and rodent reservoir samples(Andersen, et al., Cell Volume 162, Issue 4, p 738-750, 13 Aug. 2015).Andersen et al. show that whereas the 2013-2015 EVD epidemic is fueledby human-to-human transmissions, LASV infections mainly result fromreservoir-to-human infections. Andersen et al. elucidated the spread ofLASV across West Africa and show that this migration was accompanied bychanges in LASV genome abundance, fatality rates, codon adaptation, andtranslational efficiency. The method may further comprisephylogenetically comparing a first pathogen sequence to a secondpathogen sequence, and determining whether there is a phylogenetic linkbetween the first and second pathogen sequences. The second pathogensequence may be an earlier reference sequence. If there is aphylogenetic link, the method may further comprise rooting the phylogenyof the first pathogen sequence to the second pathogen sequence. Thus, itis possible to construct the lineage of the first pathogen sequence.(Park, et al., 2015).

The method may further comprise determining whether the mutations aredeleterious or adaptive. Deleterious mutations are indicative oftransmission-impaired viruses and dead-end infections, thus normallyonly present in an individual subject. Mutations unique to oneindividual subject are those that occur on the external branches of thephylogenetic tree, whereas internal branch mutations are those presentin multiple samples (i.e. in multiple subjects). Higher rate ofnonsynonymous substitution is a characteristic of external branches ofthe phylogenetic tree (Park, et al., 2015).

In internal branches of the phylogenetic tree, selection has had moreopportunity to filter out deleterious mutants. Internal branches, bydefinition, have produced multiple descendent lineages and are thus lesslikely to include mutations with fitness costs. Thus, lower rate ofnonsynonymous substitution is indicative of internal branches (Park, etal., 2015).

Synonymous mutations, which likely have less impact on fitness, occurredat more comparable frequencies on internal and external branches (Park,et al., 2015).

By analyzing the sequenced target sequence, such as viral genomes, it ispossible to discover the mechanisms responsible for the severity of theepidemic episode such as during the 2014 Ebola outbreak. For example,Gire et al. made a phylogenetic comparison of the genomes of the 2014outbreak to all 20 genomes from earlier outbreaks suggests that the 2014West African virus likely spread from central Africa within the pastdecade. Rooting the phylogeny using divergence from other ebolavirusgenomes was problematic (6, 13). However, rooting the tree on the oldestoutbreak revealed a strong correlation between sample date androot-to-tip distance, with a substitution rate of 8×10-4 per site peryear (13). This suggests that the lineages of the three most recentoutbreaks all diverged from a common ancestor at roughly the same time,around 2004, which supports the hypothesis that each outbreak representsan independent zoonotic event from the same genetically diverse viralpopulation in its natural reservoir. They also found out that the 2014EBOV outbreak might be caused by a single transmission from the naturalreservoir, followed by human-to-human transmission during the outbreak.Their results also suggested that the epidemic episode in Sierra Leonmight stem from the introduction of two genetically distinct virusesfrom Guinea around the same time (Gire, et al., 2014).

It has been also possible to determine how the Lassa virus spread outfrom its origin point, in particular thanks to human-to-humantransmission and even retrace the history of this spread 400 years back(Andersen, et al., Cell 162(4):738-50, 2015).

In relation to the work needed during the 2013-2015 EBOV outbreak andthe difficulties encountered by the medical staff at the site of theoutbreak, and more generally, the method of the invention makes itpossible to carry out sequencing using fewer selected probes such thatsequencing can be accelerated, thus shortening the time needed fromsample taking to results procurement. Further, kits and systems can bedesigned to be usable on the field so that diagnostics of a patient canbe readily performed without need to send or ship samples to anotherpart of the country or the world.

In any method described above, sequencing the target sequence orfragment thereof may be used any of the sequencing processes describedabove. Further, sequencing the target sequence or fragment thereof maybe a near-real-time sequencing. Sequencing the target sequence orfragment thereof may be carried out according to previously describedmethods (Experimental Procedures: Matranga et al., 2014; and Gire, etal., 2014). Sequencing the target sequence or fragment thereof maycomprise parallel sequencing of a plurality of target sequences.Sequencing the target sequence or fragment thereof may comprise Illuminasequencing.

Analyzing the target sequence or fragment thereof that hybridizes to oneor more of the selected probes may be an identifying analysis, whereinhybridization of a selected probe to the target sequence or a fragmentthereof indicates the presence of the target sequence within the sample.

Currently, primary diagnostics are based on the symptoms a patient has.However, various diseases may share identical symptoms so thatdiagnostics rely much on statistics. For example, malaria triggersflu-like symptoms: headache, fever, shivering, joint pain, vomiting,hemolytic anemia, jaundice, hemoglobin in the urine, retinal damage, andconvulsions. These symptoms are also common for septicemia,gastroenteritis, and viral diseases. Amongst the latter, Ebolahemorrhagic fever has the following symptoms fever, sore throat,muscular pain, headaches, vomiting, diarrhea, rash, decreased functionof the liver and kidneys, internal and external hemorrhage.

When a patient is presented to a medical unit, for example in tropicalAfrica, basic diagnostics will conclude to malaria becausestatistically, malaria is the most probable disease within that regionof Africa. The patient is consequently treated for malaria although thepatient might not actually have contracted the disease and the patientends up not being correctly treated. This lack of correct treatment canbe life-threatening especially when the disease the patient contractedpresents a rapid evolution. It might be too late before the medicalstaff realizes that the treatment given to the patient is ineffectiveand comes to the correct diagnostics and administers the adequatetreatment to the patient.

The method of the invention provides a solution to this situation.Indeed, because the number of guide RNAs can be dramatically reduced,this makes it possible to provide on a single chip selected probesdivided into groups, each group being specific to one disease, such thata plurality of diseases, e.g. viral infection, can be diagnosed at thesame time. Thanks to the invention, more than 3 diseases can bediagnosed on a single chip, preferably more than 4, 5, 6, 7, 8, 9, 10,11, 12, 13, 14, 15, 16, 17, 18, 19, 20 diseases at the same time,preferably the diseases that most commonly occur within the populationof a given geographical area. Since each group of selected probes isspecific to one of the diagnosed diseases, a more accurate diagnosis canbe performed, thus diminishing the risk of administering the wrongtreatment to the patient.

In other cases, a disease such as a viral infection may occur withoutany symptoms, or had caused symptoms but they faded out before thepatient is presented to the medical staff. In such cases, either thepatient does not seek any medical assistance or the diagnostics iscomplicated due to the absence of symptoms on the day of thepresentation.

The present invention may also be used in concert with other methods ofdiagnosing disease, identifying pathogens and optimizing treatment basedupon detection of nucleic acids, such as mRNA in crude, non-purifiedsamples.

The method of the invention also provides a powerful tool to addressthis situation. Indeed, since a plurality of groups of selected guideRNAs, each group being specific to one of the most common diseases thatoccur within the population of the given area, are comprised within asingle diagnostic, the medical staff only need to contact a biologicalsample taken from the patient with the chip. Reading the chip revealsthe diseases the patient has contracted.

In some cases, the patient is presented to the medical staff fordiagnostics of particular symptoms. The method of the invention makes itpossible not only to identify which disease causes these symptoms but atthe same time determine whether the patient suffers from another diseasehe was not aware of.

This information might be of utmost importance when searching for themechanisms of an outbreak. Indeed, groups of patients with identicalviruses also show temporal patterns suggesting a subject-to-subjecttransmission links.

Screening Microbial Genetic Perturbations

In certain example embodiments, the CRISPR systems disclosed herein maybe used to screen microbial genetic perturbations. Such methods may beuseful, for example to map out microbial pathways and functionalnetworks. Microbial cells may be genetically modified and then screenedunder different experimental conditions. As described above, theembodiments disclosed herein can screen for multiple target molecules ina single sample, or a single target in a single individual discretevolume in a multiplex fashion. Genetically modified microbes may bemodified to include a nucleic acid barcode sequence that identifies theparticular genetic modification carried by a particular microbial cellor population of microbial cells. A barcode is s short sequence ofnucleotides (for example, DNA, RNA, or combinations thereof) that isused as an identifier. A nucleic acid barcode may have a length of 4-100nucleotides and be either single or double-stranded. Methods foridentifying cells with barcodes are known in the art. Accordingly, guideRNAs of the CRISPR effector systems described herein may be used todetect the barcode. Detection of the positive detectable signalindicates the presence of a particular genetic modification in thesample. The methods disclosed herein may be combined with other methodsfor detecting complimentary genotype or phenotypic readouts indicatingthe effect of the genetic modification under the experimental conditionstested. Genetic modifications to be screened may include, but are notlimited to a gene knock-in, a gene knock-out, inversions,translocations, transpositions, or one or more nucleotide insertions,deletions, substitutions, mutations, or addition of nucleic acidsencoding an epitope with a functional consequence such as alteringprotein stability or detection. In a similar fashion, the methodsdescribed herein may be used in synthetic biology application to screenthe functionality of specific arrangements of gene regulatory elementsand gene expression modules.

In certain example embodiments, the methods may be used to screenhypomorphs. Generation of hypomorphs and their use in identifying keybacterial functional genes and identification of new antibiotictherapeutics as disclosed in PCT/US2016/060730 entitled “MultiplexHigh-Resolution Detection of Micro-organism Strains, Related Kits,Diagnostic Methods and Screening Assays” filed Nov. 4, 2016, which isincorporated herein by reference.

The different experimental conditions may comprise exposure of themicrobial cells to different chemical agents, combinations of chemicalagents, different concentrations of chemical agents or combinations ofchemical agents, different durations of exposure to chemical agents orcombinations of chemical agents, different physical parameters, or both.In certain example embodiments the chemical agent is an antibiotic orantiviral. Different physical parameters to be screened may includedifferent temperatures, atmospheric pressures, different atmospheric andnon-atmospheric gas concentrations, different pH levels, differentculture media compositions, or a combination thereof.

Screening Environmental Samples

The methods disclosed herein may also be used to screen environmentalsamples for contaminants by detecting the presence of target nucleicacid or polypeptides. For example, in some embodiments, the inventionprovides a method of detecting microbes, comprising: exposing a CRISPRsystem as described herein to a sample; activating an RNA effectorprotein via binding of one or more guide RNAs to one or moremicrobe-specific target RNAs or one or more trigger RNAs such that adetectable positive signal is produced. The positive signal can bedetected and is indicative of the presence of one or more microbes inthe sample. In some embodiments, the CRISPR system may be on a substrateas described herein, and the substrate may be exposed to the sample. Inother embodiments, the same CRISPR system, and/or a different CRISPRsystem may be applied to multiple discrete locations on the substrate.In further embodiments, the different CRISPR system may detect adifferent microbe at each location. As described in further detailabove, a substrate may be a flexible materials substrate, for example,including, but not limited to, a paper substrate, a fabric substrate, ora flexible polymer-based substrate.

In accordance with the invention, the substrate may be exposed to thesample passively, by temporarily immersing the substrate in a fluid tobe sampled, by applying a fluid to be tested to the substrate, or bycontacting a surface to be tested with the substrate. Any means ofintroducing the sample to the substrate may be used as appropriate.

As described herein, a sample for use with the invention may be abiological or environmental sample, such as a food sample (fresh fruitsor vegetables, meats), a beverage sample, a paper surface, a fabricsurface, a metal surface, a wood surface, a plastic surface, a soilsample, a freshwater sample, a wastewater sample, a saline water sample,exposure to atmospheric air or other gas sample, or a combinationthereof. For example, household/commercial/industrial surfaces made ofany materials including, but not limited to, metal, wood, plastic,rubber, or the like, may be swabbed and tested for contaminants. Soilsamples may be tested for the presence of pathogenic bacteria orparasites, or other microbes, both for environmental purposes and/or forhuman, animal, or plant disease testing. Water samples such asfreshwater samples, wastewater samples, or saline water samples can beevaluated for cleanliness and safety, and/or potability, to detect thepresence of, for example, Cryptosporidium parvum, Giardia lamblia, orother microbial contamination. In further embodiments, a biologicalsample may be obtained from a source including, but not limited to, atissue sample, saliva, blood, plasma, sera, stool, urine, sputum,mucous, lymph, synovial fluid, cerebrospinal fluid, ascites, pleuraleffusion, seroma, pus, or swab of skin or a mucosal membrane surface. Insome particular embodiments, an environmental sample or biologicalsamples may be crude samples and/or the one or more target molecules maynot be purified or amplified from the sample prior to application of themethod. Identification of microbes may be useful and/or needed for anynumber of applications, and thus any type of sample from any sourcedeemed appropriate by one of skill in the art may be used in accordancewith the invention.

In some embodiments, Checking for food contamination by bacteria, suchas E. coli, in restaurants or other food providers; food surfaces;Testing water for pathogens like Salmonella, Campylobacter, or E. coli;also checking food quality for manufacturers and regulators to determinethe purity of meat sources; identifying air contamination with pathogenssuch as legionella; Checking whether beer is contaminated or spoiled bypathogens like Pediococcus and Lactobacillus; contamination ofpasteurized or un-pasteurized cheese by bacteria or fungi duringmanufacture.

A microbe in accordance with the invention may be a pathogenic microbeor a microbe that results in food or consumable product spoilage. Apathogenic microbe may be pathogenic or otherwise undesirable to humans,animals, or plants. For human or animal purposes, a microbe may cause adisease or result in illness. Animal or veterinary applications of thepresent invention may identify animals infected with a microbe. Forexample, the methods and systems of the invention may identify companionanimals with pathogens including, but not limited to, kennel cough,rabies virus, and heartworms. In other embodiments, the methods andsystems of the invention may be used for parentage testing for breedingpurposes. A plant microbe may result in harm or disease to a plant,reduction in yield, or alter traits such as color, taste, consistency,odor, for food or consumable contamination purposes, a microbe mayadversely affect the taste, odor, color, consistency or other commercialproperties of the food or consumable product. In certain exampleembodiments, the microbe is a bacterial species. The bacteria may be apsychotroph, a coliform, a lactic acid bacteria, or a spore-formingbacterium. In certain example embodiments, the bacteria may be anybacterial species that causes disease or illness, or otherwise resultsin an unwanted product or trait. Bacteria in accordance with theinvention may be pathogenic to humans, animals, or plants.

Sample Types

Appropriate samples for use in the methods disclosed herein include anyconventional biological sample obtained from an organism or a partthereof, such as a plant, animal, bacteria, and the like. In particularembodiments, the biological sample is obtained from an animal subject,such as a human subject. A biological sample is any solid or fluidsample obtained from, excreted by or secreted by any living organism,including, without limitation, single celled organisms, such asbacteria, yeast, protozoans, and amoebas among others, multicellularorganisms (such as plants or animals, including samples from a healthyor apparently healthy human subject or a human patient affected by acondition or disease to be diagnosed or investigated, such as aninfection with a pathogenic microorganism, such as a pathogenic bacteriaor virus). For example, a biological sample can be a biological fluidobtained from, for example, blood, plasma, serum, urine, stool, sputum,mucous, lymph fluid, synovial fluid, bile, ascites, pleural effusion,seroma, saliva, cerebrospinal fluid, aqueous or vitreous humor, or anybodily secretion, a transudate, an exudate (for example, fluid obtainedfrom an abscess or any other site of infection or inflammation), orfluid obtained from a joint (for example, a normal joint or a jointaffected by disease, such as rheumatoid arthritis, osteoarthritis, goutor septic arthritis), or a swab of skin or mucosal membrane surface.

A sample can also be a sample obtained from any organ or tissue(including a biopsy or autopsy specimen, such as a tumor biopsy) or caninclude a cell (whether a primary cell or cultured cell) or mediumconditioned by any cell, tissue or organ. Exemplary samples include,without limitation, cells, cell lysates, blood smears, cytocentrifugepreparations, cytology smears, bodily fluids (e.g., blood, plasma,serum, saliva, sputum, urine, bronchoalveolar lavage, semen, etc.),tissue biopsies (e.g., tumor biopsies), fine-needle aspirates, and/ortissue sections (e.g., cryostat tissue sections and/or paraffin-embeddedtissue sections). In other examples, the sample includes circulatingtumor cells (which can be identified by cell surface markers). Inparticular examples, samples are used directly (e.g., fresh or frozen),or can be manipulated prior to use, for example, by fixation (e.g.,using formalin) and/or embedding in wax (such as formalin-fixedparaffin-embedded (FFPE) tissue samples). It will be appreciated thatany method of obtaining tissue from a subject can be utilized, and thatthe selection of the method used will depend upon various factors suchas the type of tissue, age of the subject, or procedures available tothe practitioner. Standard techniques for acquisition of such samplesare available in the art. See, for example Schluger et al., J. Exp. Med.176:1327-33 (1992); Bigby et al., Am. Rev. Respir. Dis. 133:515-18(1986); Kovacs et al., NEJM 318:589-93 (1988); and Ognibene et al., Am.Rev. Respir. Dis. 129:929-32 (1984).

In other embodiments, a sample may be an environmental sample, such aswater, soil, or a surface such as industrial or medical surface. In someembodiments, methods such as disclosed in US patent publication No.2013/0190196 may be applied for detection of nucleic acid signatures,specifically RNA levels, directly from crude cellular samples with ahigh degree of sensitivity and specificity. Sequences specific to eachpathogen of interest may be identified or selected by comparing thecoding sequences from the pathogen of interest to all coding sequencesin other organisms by BLAST software.

Several embodiments of the present disclosure involve the use ofprocedures and approaches known in the art to successfully fractionateclinical blood samples. See, e.g. the procedure described in Han WeiHour et al., Microfluidic Devices for Blood Fractionation, Micromachines2011, 2, 319-343; Ali Asgar S. Bhagat et al., Dean Flow Fractionation(DFF) Isolation of Circulating Tumor Cells (CTCs) from Blood, 15thInternational Conference on Miniaturized Systems for Chemistry and LifeSciences, Oct. 2-6, 2011, Seattle, Wash.; and International PatentPublication No. WO2011109762, the disclosures of which are hereinincorporated by reference in their entirety. Blood samples are commonlyexpanded in culture to increase sample size for testing purposes. Insome embodiments of the present invention, blood or other biologicalsamples may be used in methods as described herein without the need forexpansion in culture.

Further, several embodiments of the present disclosure involve the useof procedures and approaches known in the art to successfully isolatepathogens from whole blood using spiral microchannel, as described inHan Wei Hour et al., Pathogen Isolation from Whole Blood Using SpiralMicrochannel, Case No. 15995JR, Massachusetts Institute of Technology,manuscript in preparation, the disclosure of which is hereinincorporated by reference in its entirety.

Owing to the increased sensitivity of the embodiments disclosed herein,in certain example embodiments, the assays and methods may be run oncrude samples or samples where the target molecules to be detected arenot further fractionated or purified from the sample.

Example Microbes

The embodiment disclosed herein may be used to detect a number ofdifferent microbes. The term microbe as used herein includes bacteria,fungus, protozoa, parasites and viruses.

Bacteria

The following provides an example list of the types of microbes thatmight be detected using the embodiments disclosed herein. In certainexample embodiments, the microbe is a bacterium. Examples of bacteriathat can be detected in accordance with the disclosed methods includewithout limitation any one or more of (or any combination of)Acinetobacter baumanii, Actinobacillus sp., Actinomycetes, Actinomycessp. (such as Actinomyces israelii and Actinomyces naeslundii), Aeromonassp. (such as Aeromonas hydrophila, Aeromonas veronii biovar sobria(Aeromonas sobria), and Aeromonas caviae), Anaplasma phagocytophilum,Anaplasma marginate Alcaligenes xylosoxidans, Acinetobacter baumanii,Actinobacillus actinomycetemcomitans, Bacillus sp. (such as Bacillusanthracis, Bacillus cereus, Bacillus subtilis, Bacillus thuringiensis,and Bacillus stearothermophilus), Bacteroides sp. (such as BacteroidesBartonella sp. (such as Bartonella bacilliformis and Bartonellahenselae, Bifidobacterium sp., Bordetella sp. (such as Bordetellapertussis, Bordetella parapertussis, and Bordetella bronchiseptica),Borrelia sp. (such as Borrelia recurrentis, and Borrelia burgdorferi),Brucella sp. (such as Brucella abortus, Brucella canis, Brucellamelintensis and Brucella suis), Burkholderia sp. (such as Burkholderiapseudomallei and Burkholderia cepacia), Campylobacter sp. (such asCampylobacter jejuni, Campylobacter coli, Campylobacter lari andCampylobacter fetus), Capnocytophaga sp., Cardiobacterium hominis,Chlamydia trachomatis, Chlamydophila pneumoniae, Chlamydophila psittaci,Citrobacter sp. Coxiella burnetii, Corynebacterium sp. (such as,Corynebacterium diphtheriae, Corynebacterium jeikeum andCorynebacterium), Clostridium sp. (such as Clostridium perfringens,Clostridium difficile, Clostridium botulinum and Clostridium tetani),Eikenella corrodens, Enterobacter sp. (such as Enterobacter aerogenes,Enterobacter agglomerans, Enterobacter cloacae and Escherichia coli,including opportunistic Escherichia coli, such as enterotoxigenic E.coli, enteroinvasive E. coli, enteropathogenic E. coli,enterohemorrhagic E. coli, enteroaggregative E. coli and uropathogenicE. coli) Enterococcus sp. (such as Enterococcus faecalis andEnterococcus faecium) Ehrlichia sp. (such as Ehrlichia chafeensia andEhrlichia canis), Epidermophyton floccosum, Erysipelothrixrhusiopathiae, Eubacterium sp., Francisella tularensis, Fusobacteriumnucleatum, Gardnerella vaginalis, Gemella morbillorum, Haemophilus sp.(such as Haemophilus influenzae, Haemophilus ducreyi, Haemophilusaegyptius, Haemophilus parainfluenzae, Haemophilus haemolyticus andHaemophilus parahaemolyticus, Helicobacter sp. (such as Helicobacterpylori, Helicobacter cinaedi and Helicobacter fennelliae), Kingellakingii, Klebsiella sp. (such as Klebsiella pneumoniae, Klebsiellagranulomatis and Klebsiella oxytoca), Lactobacillus sp., Listeriamonocytogenes, Leptospira interrogans, Legionella pneumophila,Leptospira interrogans, Peptostreptococcus sp., Mannheimia hemolytica,Microsporum canis, Moraxella catarrhalis, Morganella sp., Mobiluncussp., Micrococcus sp., Mycobacterium sp. (such as Mycobacterium leprae,Mycobacterium tuberculosis, Mycobacterium paratuberculosis,Mycobacterium intracellulare, Mycobacterium avium, Mycobacterium bovis,and Mycobacterium marinum), Mycoplasm sp. (such as Mycoplasmapneumoniae, Mycoplasma hominis, and Mycoplasma genitalium), Nocardia sp.(such as Nocardia asteroides, Nocardia cyriacigeorgica and Nocardiabrasiliensis), Neisseria sp. (such as Neisseria gonorrhoeae andNeisseria meningitidis), Pasteurella multocida, Pityrosporum orbiculare(Malassezia furfur), Plesiomonas shigelloides. Prevotella sp.,Porphyromonas sp., Prevotella melaninogenica, Proteus sp. (such asProteus vulgaris and Proteus mirabilis), Providencia sp. (such asProvidencia alcalifaciens, Providencia rettgeri and Providenciastuartii), Pseudomonas aeruginosa, Propionibacterium acnes, Rhodococcusequi, Rickettsia sp. (such as Rickettsia rickettsii, Rickettsia akariand Rickettsia prowazekii, Orientia tsutsugamushi (formerly: Rickettsiatsutsugamushi) and Rickettsia typhi), Rhodococcus sp., Serratiamarcescens, Stenotrophomonas maltophilia, Salmonella sp. (such asSalmonella enterica, Salmonella typhi, Salmonella paratyphi, Salmonellaenteritidis, Salmonella cholerasuis and Salmonella typhimurium),Serratia sp. (such as Serratia marcesans and Serratia liquifaciens),Shigella sp. (such as Shigella dysenteriae, Shigella flexneri, Shigellaboydii and Shigella sonnei), Staphylococcus sp. (such as Staphylococcusaureus, Staphylococcus epidermidis, Staphylococcus hemolyticus,Staphylococcus saprophyticus), Streptococcus sp. (such as Streptococcuspneumoniae (for example chloramphenicol-resistant serotype 4Streptococcus pneumoniae, spectinomycin-resistant serotype 6BStreptococcus pneumoniae, streptomycin-resistant serotype 9VStreptococcus pneumoniae, erythromycin-resistant serotype 14Streptococcus pneumoniae, optochin-resistant serotype 14 Streptococcuspneumoniae, rifampicin-resistant serotype 18C Streptococcus pneumoniae,tetracycline-resistant serotype 19F Streptococcus pneumoniae,penicillin-resistant serotype 19F Streptococcus pneumoniae, andtrimethoprim-resistant serotype 23F Streptococcus pneumoniae,chloramphenicol-resistant serotype 4 Streptococcus pneumoniae,spectinomycin-resistant serotype 6B Streptococcus pneumoniae,streptomycin-resistant serotype 9V Streptococcus pneumoniae,optochin-resistant serotype 14 Streptococcus pneumoniae,rifampicin-resistant serotype 18C Streptococcus pneumoniae,penicillin-resistant serotype 19F Streptococcus pneumoniae, ortrimethoprim-resistant serotype 23F Streptococcus pneumoniae),Streptococcus agalactiae, Streptococcus mutans, Streptococcus pyogenes,Group A streptococci, Streptococcus pyogenes, Group B streptococci,Streptococcus agalactiae, Group C streptococci, Streptococcus anginosus,Streptococcus equismilis, Group D streptococci, Streptococcus bovis,Group F streptococci, and Streptococcus anginosus Group G streptococci),Spirillum minus, Streptobacillus moniliformi, Treponema sp. (such asTreponema carateum, Treponema petenue, Treponema pallidum and Treponemaendemicum, Trichophyton rubrum, T. mentagrophytes, Tropheryma whippelii,Ureaplasma urealyticum, Veillonella sp., Vibrio sp. (such as Vibriocholerae, Vibrio parahemolyticus, Vibrio vulnificus, Vibrioparahaemolyticus, Vibrio vulnificus, Vibrio alginolyticus, Vibriomimicus, Vibrio hollisae, Vibrio fluvialis, Vibrio metchnikovii, Vibriodamsela and Vibrio furnisii), Yersinia sp. (such as Yersiniaenterocolitica, Yersinia pestis, and Yersinia pseudotuberculosis) andXanthomonas maltophilia among others.

Fungi

In certain example embodiments, the microbe is a fungus or a fungalspecies. Examples of fungi that can be detected in accordance with thedisclosed methods include without limitation any one or more of (or anycombination of), Aspergillus, Blastomyces, Candidiasis,Coccidiodomycosis, Cryptococcus neoformans, Cryptococcus gatti, sp.Histoplasma sp. (such as Histoplasma capsulatum), Pneumocystis sp. (suchas Pneumocystis jirovecii), Stachybotrys (such as Stachybotryschartarum), Mucroymcosis, Sporothrix, fungal eye infections ringworm,Exserohilum, Cladosporium.

In certain example embodiments, the fungus is a yeast. Examples of yeastthat can be detected in accordance with disclosed methods includewithout limitation one or more of (or any combination of), Aspergillusspecies (such as Aspergillus fumigatus, Aspergillus flavus andAspergillus clavatus), Cryptococcus sp. (such as Cryptococcusneoformans, Cryptococcus gattii, Cryptococcus laurentii and Cryptococcusalbidus), a Geotrichum species, a Saccharomyces species, a Hansenulaspecies, a Candida species (such as Candida albicans), a Kluyveromycesspecies, a Debaryomyces species, a Pichia species, or combinationthereof. In certain example embodiments, the fungi is a mold. Examplemolds include, but are not limited to, a Penicillium species, aCladosporium species, a Byssochlamys species, or a combination thereof.

Protozoa

In certain example embodiments, the microbe is a protozoa. Examples ofprotozoa that can be detected in accordance with the disclosed methodsand devices include without limitation any one or more of (or anycombination of), Euglenozoa, Heterolobosea, Diplomonadida, Amoebozoa,Blastocystic, and Apicomplexa. Example Euglenoza include, but are notlimited to, Trypanosoma cruzi (Chagas disease), T. brucei gambiense, T.brucei rhodesiense, Leishmania braziliensis, L. infantum, L. mexicana,L. major, L. tropica, and L. donovani. Example Heterolobosea include,but are not limited to, Naegleria fowleri. Example Diplomonadidsinclude, but are not limited to, Giardia intestinalis (G. lamblia, G.duodenalis). Example Amoebozoa include, but are not limited to,Acanthamoeba castellanii, Balamuthia madrillaris, Entamoeba histolytica.Example Blastocysts include, but are not limited to, Blastocystichominis. Example Apicomplexa include, but are not limited to, Babesiamicroti, Cryptosporidium parvum, Cyclospora cayetanensis, Plasmodiumfalciparum, P. vivax, P. ovale, P. malariae, and Toxoplasma gondii.

Parasites

In certain example embodiments, the microbe is a parasite. Examples ofparasites that can be detected in accordance with disclosed methodsinclude without limitation one or more of (or any combination of), anOnchocerca species and a Plasmodium species.

Viruses

In certain example embodiments, the systems, devices, and methods,disclosed herein are directed to detecting viruses in a sample. Theembodiments disclosed herein may be used to detect viral infection (e.g.of a subject or plant), or determination of a viral strain, includingviral strains that differ by a single nucleotide polymorphism. The virusmay be a DNA virus, a RNA virus, or a retrovirus. Non-limiting exampleof viruses useful with the present invention include, but are notlimited to Ebola, measles, SARS, Chikungunya, hepatitis, Marburg, yellowfever, MERS, Dengue, Lassa, influenza, rhabdovirus or HIV. A hepatitisvirus may include hepatitis A, hepatitis B, or hepatitis C. An influenzavirus may include, for example, influenza A or influenza B. An HIV mayinclude HIV 1 or HIV 2. In certain example embodiments, the viralsequence may be a human respiratory syncytial virus, Sudan ebola virus,Bundibugyo virus, Tai Forest ebola virus, Reston ebola virus, Achimota,Aedes flavivirus, Aguacate virus, Akabane virus, Alethinophidreptarenavirus, Allpahuayo mammarenavirus, Amapari mmarenavirus, Andesvirus, Apoi virus, Aravan virus, Aroa virus, Arumwot virus, Atlanticsalmon paramyxovirus, Australian bat lyssavirus, Avian bornavirus, Avianmetapneumovirus, Avian paramyxoviruses, penguin or FalklandIslandsvirus, BK polyomavirus, Bagaza virus, Banna virus, Batherpesvirus, Bat sapovirus, Bear Canon mammarenavirus, Beilong virus,Betacoronavirus, Betapapillomavirus 1-6, Bhanja virus, Bokeloh batlyssavirus, Borna disease virus, Bourbon virus, Bovine hepacivirus,Bovine parainfluenza virus 3, Bovine respiratory syncytial virus,Brazoran virus, Bunyamwera virus, Caliciviridae virus. Californiaencephalitis virus, Candiru virus, Canine distemper virus, Caninepneumovirus, Cedar virus, Cell fusing agent virus, Cetaceanmorbillivirus, Chandipura virus, Chaoyang virus, Chapare mammarenavirus,Chikungunya virus, Colobus monkey papillomavirus, Colorado tick fevervirus, Cowpox virus, Crimean-Congo hemorrhagic fever virus, Culexflavivirus, Cupixi mammarenavirus, Dengue virus, Dobrava-Belgrade virus,Donggang virus, Dugbe virus, Duvenhage virus, Eastern equineencephalitis virus, Entebbe bat virus, Enterovirus A-D, European batlyssavirus 1-2, Eyach virus, Feline morbillivirus, Fer-de-Lanceparamyxovirus, Fitzroy River virus, Flaviviridae virus, Flexalmammarenavirus, GB virus C, Gairo virus, Gemycircularvirus, Gooseparamyxovirus SF02, Great Island virus, Guanarito mammarenavirus,Hantaan virus, Hantavirus Z10, Heartland virus, Hendra virus, HepatitisA/B/C/E, Hepatitis delta virus, Human bocavirus, Human coronavirus,Human endogenous retrovirus K, Human enteric coronavirus, Humangenital-associated circular DNA virus-1, Human herpesvirus 1-8, Humanimmunodeficiency virus 1/2, Human mastadenovirus A-G, Humanpapillomavirus, Human parainfluenza virus 1-4, Human paraechovirus,Human picornavirus, Human smacovirus, Ikoma lyssavirus, Ilheus virus,Influenza A-C, Ippy mammarenavirus, Irkut virus, J-virus, JCpolyomavirus, Japanese encephalitis virus, Junin mammarenavirus, KIpolyomavirus, Kadipiro virus, Kamiti River virus, Kedougou virus, Khujand virus, Kokobera virus, Kyasanur forest disease virus, Lagos batvirus, Langat virus, Lassa mammarenavirus, Latino mammarenavirus,Leopards Hill virus, Liao ning virus, Ljungan virus, Lloviu virus,Louping ill virus, Lujo mammarenavirus, Luna mammarenavirus, Lunk virus,Lymphocytic choriomeningitis mammarenavirus, Lyssavirus Ozernoe,MSSI2\0.225 virus, Machupo mammarenavirus, Mamastrovirus 1, Manzanillavirus, Mapuera virus, Marburg virus, Mayaro virus, Measles virus,Menangle virus, Mercadeo virus, Merkel cell polyomavirus, Middle Eastrespiratory syndrome coronavirus, Mobala mammarenavirus, Modoc virus,Moijang virus, Mokolo virus, Monkeypox virus, Montana myotisleukoenchalitis virus, Mopeia lassa virus reassortant 29, Mopeiamammarenavirus, Morogoro virus, Mossman virus, Mumps virus, Murinepneumonia virus, Murray Valley encephalitis virus, Nariva virus,Newcastle disease virus, Nipah virus, Norwalk virus, Norway rathepacivirus, Ntaya virus, O'nyong-nyong virus, Oliveros mammarenavirus,Omsk hemorrhagic fever virus, Oropouche virus, Parainfluenza virus 5,Parana mammarenavirus, Parramatta River virus,Peste-des-petits-ruminants virus, Pichande mammarenavirus,Picornaviridae virus, Pirital mammarenavirus, Piscihepevirus A, Porcineparainfluenza virus 1, porcine rubulavirus, Powassan virus, PrimateT-lymphotropic virus 1-2, Primate erythroparvovirus 1, Punta Toro virus,Puumala virus, Quang Binh virus, Rabies virus, Razdan virus, Reptilebornavirus 1, Rhinovirus A-B, Rift Valley fever virus, Rinderpest virus,Rio Bravo virus, Rodent Torque Teno virus, Rodent hepacivirus, RossRiver virus, Rotavirus A-I, Royal Farm virus, Rubella virus, Sabiamammarenavirus, Salem virus, Sandfly fever Naples virus, Sandfly feverSicilian virus, Sapporo virus, Sathuperi virus, Seal anellovirus,Semliki Forest virus, Sendai virus, Seoul virus, Sepik virus, Severeacute respiratory syndrome-related coronavirus, Severe fever withthrombocytopenia syndrome virus, Shamonda virus, Shimoni bat virus,Shuni virus, Simbu virus, Simian torque teno virus, Simian virus 40-41,Sin Nombre virus, Sindbis virus, Small anellovirus, Sosuga virus,Spanish goat encephalitis virus, Spondweni virus, St. Louis encephalitisvirus, Sunshine virus, TTV-like mini virus, Tacaribe mammarenavirus,Taila virus, Tamana bat virus, Tamiami mammarenavirus, Tembusu virus,Thogoto virus, Thottapalayam virus, Tick-borne encephalitis virus,Tioman virus, Togaviridae virus, Torque teno canis virus, Torque tenodouroucouli virus, Torque teno felis virus, Torque teno midi virus,Torque teno sus virus, Torque teno tamarin virus, Torque teno virus,Torque teno zalophus virus, Tuhoko virus, Tula virus, Tupaiaparamyxovirus, Usutu virus, Uukuniemi virus, Vaccinia virus, Variolavirus, Venezuelan equine encephalitis virus, Vesicular stomatitisIndiana virus, WU Polyomavirus, Wesselsbron virus, West Caucasian batvirus, West Nile virus, Western equine encephalitis virus, WhitewaterArroyo mammarenavirus, Yellow fever virus, Yokose virus, Yug Bogdanovacvirus, Zaire ebolavirus, Zika virus, or Zygosaccharomyces bailii virus Zviral sequence. Examples of RNA viruses that may be detected include oneor more of (or any combination of) Coronaviridae virus, a Picornaviridaevirus, a Caliciviridae virus, a Flaviviridae virus, a Togaviridae virus,a Bornaviridae, a Filoviridae, a Paramyxoviridae, a Pneumoviridae, aRhabdoviridae, an Arenaviridae, a Bunyaviridae, an Orthomyxoviridae, ora Deltavirus. In certain example embodiments, the virus is Coronavirus,SARS, Poliovirus, Rhinovirus, Hepatitis A, Norwalk virus, Yellow fevervirus, West Nile virus, Hepatitis C virus, Dengue fever virus, Zikavirus, Rubella virus, Ross River virus, Sindbis virus, Chikungunyavirus, Borna disease virus, Ebola virus, Marburg virus, Measles virus,Mumps virus, Nipah virus, Hendra virus, Newcastle disease virus, Humanrespiratory syncytial virus, Rabies virus, Lassa virus, Hantavirus,Crimean-Congo hemorrhagic fever virus, Influenza, or Hepatitis D virus.

In certain example embodiments, the virus may be a plant virus selectedfrom the group comprising Tobacco mosaic virus (TMV), Tomato spottedwilt virus (TSWV), Cucumber mosaic virus (CMV), Potato virus Y (PVY),the RT virus Cauliflower mosaic virus (CaMV), Plum pox virus (PPV),Brome mosaic virus (BMV), Potato virus X (PVX), Citrus tristeza virus(CTV), Barley yellow dwarf virus (BYDV), Potato leafroll virus (PLRV),Tomato bushy stunt virus (TBSV), rice tungro spherical virus (RTSV),rice yellow mottle virus (RYMV), rice hoja blanca virus (RHBV), maizerayado fino virus (MRFV), maize dwarf mosaic virus (MDMV), sugarcanemosaic virus (SCMV), Sweet potato feathery mottle virus (SPFMV), sweetpotato sunken vein closterovirus (SPSVV), Grapevine fanleaf virus(GFLV), Grapevine virus A (GVA), Grapevine virus B (GVB), Grapevinefleck virus (GFkV), Grapevine leafroll-associated virus-1, -2, and -3,(GLRaV-1, -2, and -3), Arabis mosaic virus (ArMV), or Rupestris stempitting-associated virus (RSPaV). In a preferred embodiment, the targetRNA molecule is part of said pathogen or transcribed from a DNA moleculeof said pathogen. For example, the target sequence may be comprised inthe genome of an RNA virus. It is further preferred that CRISPR effectorprotein hydrolyzes said target RNA molecule of said pathogen in saidplant if said pathogen infects or has infected said plant. It is thuspreferred that the CRISPR system is capable of cleaving the target RNAmolecule from the plant pathogen both when the CRISPR system (or partsneeded for its completion) is applied therapeutically, i.e. afterinfection has occurred or prophylactically, i.e. before infection hasoccurred.

In certain example embodiments, the virus may be a retrovirus. Exampleretroviruses that may be detected using the embodiments disclosed hereininclude one or more of or any combination of viruses of the GenusAlpharetrovirus, Betaretrovirus, Gammaretrovirus, Deltaretrovirus,Epsilonretrovirus, Lentivirus, Spumavirus, or the Family Metaviridae,Pseudoviridae, and Retroviridae (including HIV), Hepadnaviridae(including Hepatitis B virus), and Caulimoviridae (including Cauliflowermosaic virus).

In certain example embodiments, the virus is a DNA virus. Example DNAviruses that may be detected using the embodiments disclosed hereininclude one or more of (or any combination of) viruses from the FamilyMyoviridae, Podoviridae, Siphoviridae, Alloherpesviridae, Herpesviridae(including human herpes virus, and Varicella Zorter virus),Malocoherpesviridae, Lipothrixviridae, Rudiviridae, Adenoviridae,Ampullaviridae, Ascoviridae, Asfarviridae (including African swine fevervirus), Baculoviridae, Cicaudaviridae, Clavaviridae, Corticoviridae,Fuselloviridae, Globuloviridae, Guttaviridae, Hytrosaviridae,Iridoviridae, Maseilleviridae, Mimiviridae, Nudiviridae, Nimaviridae,Pandoraviridae, Papillomaviridae, Phycodnaviridae, Plasmaviridae,Polydnaviruses, Polyomaviridae (including Simian virus 40, JC virus, BKvirus), Poxviridae (including Cowpox and smallpox), Sphaerolipoviridae,Tectiviridae, Turriviridae, Dinodnavirus, Salterprovirus, Rhizidovirus,among others. In some embodiments, a method of diagnosing aspecies-specific bacterial infection in a subject suspected of having abacterial infection is described as obtaining a sample comprisingbacterial ribosomal ribonucleic acid from the subject; contacting thesample with one or more of the probes described, and detectinghybridization between the bacterial ribosomal ribonucleic acid sequencepresent in the sample and the probe, wherein the detection ofhybridization indicates that the subject is infected with Escherichiacoli, Klebsiella pneumoniae, Pseudomonas aeruginosa, Staphylococcusaureus, Acinetobacter baumannii, Candida albicans, Enterobacter cloacae,Enterococcus faecalis, Enterococcus faecium, Proteus mirabilis,Staphylococcus agalactiae, or Staphylococcus maltophilia or acombination thereof.

Malaria Detection and Monitoring

Malaria is a mosquito-borne pathology caused by Plasmodium parasites.The parasites are spread to people through the bites of infected femaleAnopheles mosquitoes. Five Plasmodium species cause malaria in humans:Plasmodium falciparum, Plasmodium vivax, Plasmodium ovale, Plasmodiummalariae, and Plasmodium knowlesi. Among them, according to the WorldHealth Organization (WHO), Plasmodium falciparum and Plasmodium vivaxare responsible for the greatest threat. P. falciparum is the mostprevalent malaria parasite on the African continent and is responsiblefor most malaria-related deaths globally. P. vivax is the dominantmalaria parasite in most countries outside of sub-Saharan Africa.

In 2015, 91 countries and areas had ongoing malaria transmission.According to the latest WHO estimates, there were 212 million cases ofmalaria in 2015 and 429 000 deaths. In areas with high transmission ofmalaria, children under 5 are particularly susceptible to infection,illness and death; more than two thirds (70%) of all malaria deathsoccur in this age group. Between 2010 and 2015, the under-5 malariadeath rate fell by 29% globally. However malaria remains a major killerof children under five years old, taking the life of a child every twominutes.

As described by the WHO, malaria is an acute febrile illness. In anon-immune individual, symptoms appear 7 days or more after theinfective mosquito bite. The first symptoms—fever, headache, chills andvomiting—may be mild and difficult to recognize as malaria, however, ifnot treated within 24 hours, P. falciparum malaria can progress tosevere illness, often leading to death.

Children with severe malaria frequently develop one or more of thefollowing symptoms: severe anemia, respiratory distress in relation tometabolic acidosis, or cerebral malaria. In adults, multi-organinvolvement is also frequent. In malaria endemic areas, people maydevelop partial immunity, allowing asymptomatic infections to occur.

The development of rapid and efficient diagnostic tests is of highrelevance for public health. Indeed, early diagnosis and treatment ofmalaria not only reduces disease and prevents deaths but alsocontributes to reducing malaria transmission. According to the WHOrecommendations, all cases of suspected malaria should be confirmedusing parasite-based diagnostic testing (notably using a rapiddiagnostic test) before administering treatment (see “WHO Guidelines forthe treatment of malaria”, third edition, published in April 2015).

Resistance to antimalarial therapies represents a critical healthproblem which drastically reduces therapeutic strategies. Indeed, asreported on the WHO website, resistance of P. falciparum to previousgenerations of medicines, such as chloroquine andsulfadoxine/pyrimethamine (SP), became widespread in the 1950s and1960s, undermining malaria control efforts and reversing gains in childsurvival. Thus, the WHO recommends the routine monitoring ofantimalarial drug resistance. Indeed, accurate diagnostic may avoid nonappropriate treatments and limit extension of resistance to antimalarialmedicines.

In this context the WHO Global Technical Strategy for Malaria2016-2030—adopted by the World Health Assembly in May 2015—provides atechnical framework for all malaria-endemic countries. It is intended toguide and support regional and country programs as they work towardsmalaria control and elimination. The Strategy sets ambitious butachievable global targets, including:

-   -   Reducing malaria case incidence by at least 90% by 2030.    -   Reducing malaria mortality rates by at least 90% by 2030.    -   Eliminating malaria in at least 35 countries by 2030.    -   Preventing a resurgence of malaria in all countries that are        malaria-free.

This Strategy was the result of an extensive consultative process thatspanned 2 years and involved the participation of more than 400technical experts from 70 Member States. It is based on 3 key axes:

-   -   ensuring universal access to malaria prevention, diagnosis and        treatment;    -   accelerating efforts towards elimination and attainment of        malaria-free status; and    -   transforming malaria surveillance into a core intervention.

Treatment against Plasmodium include aryl-amino alcohols such as quinineor quinine derivatives such as chloroquine, amodiaquine, mefloquine,piperaquine, lumefantrine, primaquine; lipophilic hydroxynaphthoquinoneanalog, such as atovaquone; antifolate drugs, such as the sulfa drugssulfadoxine, dapsone and pyrimethamine; proguanil; the combination ofatovaquone/proguanil; atemisins drugs; and combinations thereof.

Target sequences that are diagnostic for the presence of amosquito-borne pathogen include sequence that diagnostic for thepresence of Plasmodium, notably Plasmodia species affecting humans suchas Plasmodium falciparum, Plasmodium vivax, Plasmodium ovale, Plasmodiummalariae, and Plasmodium knowlesi, including sequences from the genomesthereof.

Target sequences that are diagnostic for monitoring drug resistance totreatment against Plasmodium, notably Plasmodia species affecting humanssuch as Plasmodium falciparum, Plasmodium vivax, Plasmodium ovale,Plasmodium malariae, and Plasmodium knowlesi.

Further target sequence include sequences include targetmolecules/nucleic acid molecules coding for proteins involved inessential biological process for the Plasmodium parasite and notablytransporter proteins, such as protein from drug/metabolite transporterfamily, the ATP-binding cassette (ABC) protein involved in substratetranslocation, such as the ABC transporter C subfamily or the Na⁺/H⁺exchanger, membrane glutathione S-transferase; proteins involved in thefolate pathway, such as the dihydropteroate synthase, the dihydrofolatereductase activity or the dihydrofolate reductase-thymidylate synthase;and proteins involved in the translocation of protons across the innermitochondrial membrane and notably the cytochrome b complex. Additionaltarget may also include the gene(s) coding for the heme polymerase.

Further target sequences include target molecules/nucleic acid moleculescoding for proteins involved in essential biological process may beselected from the P. falciparum chloroquine resistance transporter gene(pfcrt), the P. falciparum multidrug resistance transporter 1 (pfindr1),the P. falciparum multidrug resistance-associated protein gene (Pfmrp),the P. falciparum Na+/H+ exchanger gene (pfnhe), the gene coding for theP. falciparum exported protein 1, the P. falciparum Ca2+ transportingATPase 6 (pfatp6); the P. falciparum dihydropteroate synthase (pfdhps),dihydrofolate reductase activity (pfdhpr) and dihydrofolatereductase-thymidylate synthase (pfdhfr) genes, the cytochrome b gene,GTP cyclohydrolase and the Kelch13 (K13) gene as well as theirfunctional heterologous genes in other Plasmodium species.

A number of mutations, notably single point mutations, have beenidentified in the proteins which are the targets of the currenttreatments and associated with specific resistance phenotypes.Accordingly, the invention allows for the detection of variousresistance phenotypes of mosquito-borne parasites, such as plasmodium.

The invention allows to detect one or more mutation(s) and notably oneor more single nucleotide polymorphisms in target nucleicacids/molecules. Accordingly any one of the mutations below, or theircombination thereof, can be used as drug resistance marker and can bedetected according to the invention.

Single point mutations in P. falciparum K13 include the following singlepoint mutations in positions 252, 441, 446, 449, 458, 493, 539, 543,553, 561, 568, 574, 578, 580, 675, 476, 469, 481, 522, 537, 538, 579,584 and 719 and notably mutations E252Q, P441L, F4461, G449A, N458Y,Y493H, R539T, I543T, P553L, R561H, V568G, P574L, A578S, C580Y, A675V,M4761; C469Y; A481V; S522C; N5371; N537D; G538V; M5791; D584V; andH719N. These mutations are generally associated with artemisins drugsresistance phenotypes (Artemisinin and artemisinin-based combinationtherapy resistance, April 2016 WHO/HTM/GMP/2016.5).

In the P. falciparum dihydrofolate reductase (DHFR) (PfDHFR-TS,PFD0830w), important polymorphisms include mutations in positions 108,51, 59 and 164, notably 108 D, 164L, 51I and 59R which modulateresistance to pyrimethamine. Other polymorphisms also include 437G,581G, 540E, 436A and 613S which are associated with resistance tosulfadoxine. Additional observed mutations include Ser108Asn, Asn51Ile,Cys59Arg, Ile164Leu, Cys50Arg, Ile164Leu, Asn188Lys, Ser189Arg andVal213Ala, Ser108Thr and Ala16Val. Mutations Ser108Asn, Asn51Ile,Cys59Arg, Ile164Leu, Cys50Arg, Ile164Leu are notably associated withpyrimethamine based therapy and/or chloroguanine-dapsone combinationtherapy resistances. Cycloguanil resistance appears to be associatedwith the double mutations Ser108Thr and Ala16Val. Amplification of dhfrmay also be of high relevance for therapy resistance notablypyrimethamine resistance

In the P. falciparum dihydropteroate synthase (DHPS) (PfDHPS,PF08_0095), important polymorphisms include mutations in positions 436,437, 581 and 613 Ser436A1a/Phe, Ala437Gly, Lys540Glu, Ala581Gly andAla613Thr/Ser. Polymorphism in position 581 and/or 613 have also beenassociated with resistance to sulfadoxine-pyrimethamine base therapies.

In the P. falciparum chloroquine-resistance transporter (PfCRT),polymorphism in position 76, notably the mutation Lys76Thr, isassociated with resistance to chloroquine. Further polymorphisms includeCys72Ser, Met74Ile, Asn75Glu, Ala220Ser, Gln271Glu, Asn326Ser, Ile356Thrand Arg371Ile which may be associated with chloroquine resistance. PfCRTis also phosphorylated at the residues S33, S411 and T416, which mayregulate the transport activity or specificity of the protein.

In the P. falciparum multidrug-resistance transporter 1 (PfMDR1)(PFE1150w), polymorphisms in positions 86, 184, 1034, 1042, notablyAsn86Tyr, Tyr184-Phe, Ser1034Cys, Asn1042Asp and Asp1246Tyr have beenidentified and reported to influence have been reported to influencesusceptibilities to lumefantrine, artemisinin, quinine, mefloquine,halofantrine and chloroquine. Additionally, amplification of PfMDR1 isassociated with reduced susceptibility to lumefantrine, artemisinin,quinine, mefloquine, and halofantrine and deamplification of PfMDR1leads to an increase in chloroquine resistance. Amplification of pfmdr1may also be detected. The phosphorylation status of PfMDR1 is also ofhigh relevance.

In the P. falciparum multidrug-resistance associated protein (PfMRP)(gene reference PFA0590w), polymorphisms in positions 191 and/or 437,such as Y191H and A437S have been identified and associated withchloroquine resistance phenotypes.

In the P. falciparum NA+/H+ enchanger (PfNHE) (ref PF13_0019), increasedrepetition of the DNNND in microsatellite ms4670 may be a marker forquinine resistance.

Mutations altering the ubiquinol binding site of the cytochrome bprotein encoded by the cytochrome be gene (cytb, mal_mito_3) areassociated with atovaquone resistance. Mutations in positions 26, 268,276, 133 and 280 and notably Tyr26Asn, Tyr268Ser, M1331 and G280D may beassociated with atovaquone resistance.

For example in P vivax, mutations in PvMDR1, the homolog of Pf MDR1 havebeen associated with chloroquine resistance, notably polymorphism inposition 976 such as the mutation Y976F.

The above mutations are defined in terms of protein sequences. However,the skilled person is able to determine the corresponding mutations,including SNPS, to be identified as a nucleic acid target sequence.

Other identified drug-resistance markers are known in the art, forexample as described in “Susceptibility of Plasmodium falciparum toantimalarial drugs (1996-2004)”, WHO; Artemisinin and artemisinin-basedcombination therapy resistance (April 2016 WHO/HTM/GMP/2016.5);“Drug-resistant malaria: molecular mechanisms and implications forpublic health” FEBS Lett. 2011 Jun. 6; 585(11):1551-62.doi:10.1016/j.febslet.2011.04.042. Epub 2011 Apr. 23. Review. PubMedPMID: 21530510; the contents of which are herewith incorporated byreference

As to polypeptides that may be detected in accordance with the presentinvention, gene products of all genes mentioned herein may be used astargets. Correspondingly, it is contemplated that such polypeptidescould be used for species identification, typing and/or detection ofdrug resistance.

In certain example embodiments, the systems, devices, and methods,disclosed herein are directed to detecting the presence of one or moremosquito-borne parasite in a sample, such as a biological sampleobtained from a subject. In certain example embodiments, the parasitemay be selected from the species Plasmodium falciparum, Plasmodiumvivax, Plasmodium ovale, Plasmodium malariae or Plasmodium knowlesi.Accordingly, the methods disclosed herein can be adapted for use inother methods (or in combination) with other methods that require quickidentification of parasite species, monitoring the presence of parasitesand parasite forms (for example corresponding to various stages ofinfection and parasite life-cycle, such as exo-erythrocytic cycle,erythrocytic cycle, sporogonic cycle; parasite forms include merozoites,sporozoites, schizonts, gametocytes); detection of certain phenotypes(e.g. pathogen drug resistance), monitoring of disease progressionand/or outbreak, and treatment (drug) screening. Further, in the case ofmalaria, a long time may elapse following the infective bite, namely along incubation period, during which the patient does not show symptoms.Similarly, prophylactic treatments can delay the appearance of symptoms,and long asymptomatic periods can also be observed before a relapse.Such delays can easily cause misdiagnosis or delayed diagnosis, and thusimpair the effectiveness of treatment.

Because of the rapid and sensitive diagnostic capabilities of theembodiments disclosed here, detection of parasite type, down to a singlenucleotide difference, and the ability to be deployed as a POC device,the embodiments disclosed herein may be used guide therapeutic regimens,such as selection of the appropriate course of treatment. Theembodiments disclosed herein may also be used to screen environmentalsamples (mosquito population, etc.) for the presence and the typing ofthe parasite. The embodiments may also be modified to detectmosquito-borne parasites and other mosquito-borne pathogenssimultaneously. In some instances, malaria and other mosquito-bornepathogens may present initially with similar symptoms. Thus, the abilityto quickly distinguish the type of infection can guide importanttreatment decisions. Other mosquito-borne pathogens that may be detectedin conjunction with malaria include dengue, West Nile virus,chikungunya, yellow fever, filariasis, Japanese encephalitis, SaintLouis encephalitis, western equine encephalitis, eastern equineencephalitis, Venezuelan equine encephalitis, La Crosse encephalitis,and zika.

In certain example embodiments, the devices, systems, and methodsdisclosed herein may be used to distinguish multiple mosquito-borneparasite species in a sample. In certain example embodiments,identification may be based on ribosomal RNA sequences, including the18S, 16S, 23S, and 5S subunits. In certain example embodiments,identification may be based on sequences of genes that are present inmultiple copies in the genome, such as mitochondrial genes like CYTB. Incertain example embodiments, identification may be based on sequences ofgenes that are highly expressed and/or highly conserved such as GAPDH,Histone H2B, enolase, or LDH. Methods for identifying relevant rRNAsequences are disclosed in U.S. Patent Application Publication No.2017/0029872. In certain example embodiments, a set of guide RNA maydesigned to distinguish each species by a variable region that is uniqueto each species or strain. Guide RNAs may also be designed to target RNAgenes that distinguish microbes at the genus, family, order, class,phylum, kingdom levels, or a combination thereof. In certain exampleembodiments where amplification is used, a set of amplification primersmay be designed to flanking constant regions of the ribosomal RNAsequence and a guide RNA designed to distinguish each species by avariable internal region. In certain example embodiments, the primersand guide RNAs may be designed to conserved and variable regions in the16S subunit respectfully. Other genes or genomic regions that uniquelyvariable across species or a subset of species such as the RecA genefamily, RNA polymerase β subunit, may be used as well. Other suitablephylogenetic markers, and methods for identifying the same, arediscussed for example in Wu et al. arXiv:1307.8690 [q-bio.GN].

In certain example embodiments, species identification can be performedbased on genes that are present in multiple copies in the genome, suchas mitochondrial genes like CYTB. In certain example embodiments,species identification can be performed based on highly expressed and/orhighly conserved genes such as GAPDH, Histone H2B, enolase, or LDH.

In certain example embodiments, a method or diagnostic is designed toscreen mosquito-borne parasites across multiple phylogenetic and/orphenotypic levels at the same time. For example, the method ordiagnostic may comprise the use of multiple CRISPR systems withdifferent guide RNAs. A first set of guide RNAs may distinguish, forexample, between Plasmodium falciparum or Plasmodium vivax. Thesegeneral classes can be even further subdivided. For example, guide RNAscould be designed and used in the method or diagnostic that distinguishdrug-resistant strains, in general or with respect to a specific drug orcombination of drugs. A second set of guide RNA can be designed todistinguish microbes at the species level. Thus a matrix may be producedidentifying all mosquito-borne parasites species or subspecies, furtherdivided according to drug resistance. The foregoing is for examplepurposes only. Other means for classifying other types of mosquito-borneparasites are also contemplated and would follow the general structuredescribed above.

In certain example embodiments, the devices, systems and methodsdisclosed herein may be used to screen for mosquito-borne parasite genesof interest, for example drug resistance genes. Guide RNAs may bedesigned to distinguish between known genes of interest. Samples,including clinical samples, may then be screened using the embodimentsdisclosed herein for detection of one or more such genes. The ability toscreen for drug resistance at POC would have tremendous benefit inselecting an appropriate treatment regime. In certain exampleembodiments, the drug resistance genes are genes encoding proteins suchas transporter proteins, such as protein from drug/metabolitetransporter family, the ATP-binding cassette (ABC) protein involved insubstrate translocation, such as the ABC transporter C subfamily or theNa⁺/H⁺ exchanger; proteins involved in the folate pathway, such as thedihydropteroate synthase, the dihydrofolate reductase activity or thedihydrofolate reductase-thymidylate synthase; and proteins involved inthe translocation of protons across the inner mitochondrial membrane andnotably the cytochrome b complex. Additional targets may also includethe gene(s) coding for the heme polymerase. In certain exampleembodiments, the drug resistance genes are selected from the P.falciparum chloroquine resistance transporter gene (pfcrt), the P.falciparum multidrug resistance transporter 1 (pfmdr1), the P.falciparum multidrug resistance-associated protein gene (Pfmrp), the P.falciparum Na⁺/H⁺ exchanger gene (pfnhe), the P. falciparum Ca2+transporting ATPase 6 (pfatp6), the P. falciparum dihydropteroatesynthase (pfdhps), dihydrofolate reductase activity (pfdhpr) anddihydrofolate reductase-thymidylate synthase (pfdhfr) genes, thecytochrome b gene, GTP cyclohydrolase and the Kelch13 (K13) gene as wellas their functional heterologous genes in other Plasmodium species.Other identified drug-resistance markers are known in the art, forexample as described in “Susceptibility of Plasmodium falciparum toantimalarial drugs (1996-2004)”, WHO; Artemisinin and artemisinin-basedcombination therapy resistance (April 2016 WHO/HTM/GMP/2016.5);“Drug-resistant malaria: molecular mechanisms and implications forpublic health” FEBS Lett. 2011 Jun. 6; 585(11):1551-62.doi:10.1016/j.febslet.2011.04.042. Epub 2011 Apr. 23. Review. PubMedPMID: 21530510; the contents of which are herewith incorporated byreference.

In some embodiments, a CRISPR system, detection system or methods of usethereof as described herein may be used to determine the evolution of amosquito-borne parasite outbreak. The method may comprise detecting oneor more target sequences from a plurality of samples from one or moresubjects, wherein the target sequence is a sequence from amosquito-borne parasite spreading or causing the outbreaks. Such amethod may further comprise determining a pattern of mosquito-borneparasite transmission, or a mechanism involved in a disease outbreakcaused by a mosquito-borne parasite. The samples may be derived from oneor more humans, and/or be derived from one or more mosquitoes.

The pattern of pathogen transmission may comprise continued newtransmissions from the natural reservoir of the mosquito-borne parasiteor other transmissions (e.g. across mosquitoes) following a singletransmission from the natural reservoir or a mixture of both. In oneembodiment, the target sequence is preferably a sequence within themosquito-borne parasite genome or fragments thereof. In one embodiment,the pattern of the mosquito-borne parasite transmission is the earlypattern of the mosquito-borne parasite transmission, i.e. at thebeginning of the mosquito-borne parasite outbreak. Determining thepattern of the mosquito-borne parasite transmission at the beginning ofthe outbreak increases likelihood of stopping the outbreak at theearliest possible time thereby reducing the possibility of local andinternational dissemination.

Determining the pattern of the mosquito-borne parasite transmission maycomprise detecting a mosquito-borne parasite sequence according to themethods described herein. Determining the pattern of the pathogentransmission may further comprise detecting shared intra-host variationsof the mosquito-borne parasite sequence between the subjects anddetermining whether the shared intra-host variations show temporalpatterns. Patterns in observed intrahost and interhost variation provideimportant insight about transmission and epidemiology (Gire, et al.,2014).

In addition to other sample types disclosed herein, the sample may bederived from one or more mosquitoes, for example the sample may comprisemosquito saliva.

Biomarker Detection

In certain example embodiments, the systems, devices, and methodsdisclosed herein may be used for biomarker detection. For example, thesystems, devices and method disclosed herein may be used for SNPdetection and/or genotyping. The systems, devices and methods disclosedherein may be also used for the detection of any disease state ordisorder characterized by aberrant gene expression. Aberrant geneexpression includes aberration in the gene expressed, location ofexpression and level of expression. Multiple transcripts or proteinmarkers related to cardiovascular, immune disorders, and cancer amongother diseases may be detected. In certain example embodiments, theembodiments disclosed herein may be used for cell free DNA detection ofdiseases that involve lysis, such as liver fibrosis andrestrictive/obstructive lung disease. In certain example embodiments,the embodiments could be utilized for faster and more portable detectionfor pre-natal testing of cell-free DNA. The embodiments disclosed hereinmay be used for screening panels of different SNPs associated with,among others, cardiovascular health, lipid/metabolic signatures,ethnicity identification, paternity matching, human ID (e.g. matchingsuspect to a criminal database of SNP signatures). The embodimentsdisclosed herein may also be used for cell free DNA detection ofmutations related to and released from cancer tumors. The embodimentsdisclosed herein may also be used for detection of meat quality, forexample, by providing rapid detection of different animal sources in agiven meat product. Embodiments disclosed herein may also be used forthe detection of GMOs or gene editing related to DNA. As describedherein elsewhere, closely related genotypes/alleles or biomarkers (e.g.having only a single nucleotide difference in a given target sequence)may be distinguished by introduction of a synthetic mismatch in thegRNA.

In an aspect, the invention relates to a method for detecting targetnucleic acids in samples, comprising:

-   -   a. distributing a sample or set of samples into one or more        individual discrete volumes, the individual discrete volumes        comprising a CRISPR system according to the invention as        described herein;    -   b. incubating the sample or set of samples under conditions        sufficient to allow binding of the one or more guide RNAs to one        or more target molecules;    -   c. activating the CRISPR effector protein via binding of the one        or more guide RNAs to the one or more target molecules, wherein        activating the CRISPR effector protein results in modification        of the RNA-based masking construct such that a detectable        positive signal is generated; and    -   d. detecting the detectable positive signal, wherein detection        of the detectable positive signal indicates a presence of one or        more target molecules in the sample.

Biomarker Sample Types

The sensitivity of the assays described herein are well suited fordetection of target nucleic acids in a wide variety of biological sampletypes, including sample types in which the target nucleic acid is diluteor for which sample material is limited. Biomarker screening may becarried out on a number of sample types including, but not limited to,saliva, urine, blood, feces, sputum, and cerebrospinal fluid. Theembodiments disclosed herein may also be used to detect up- and/ordown-regulation of genes. For example, a s sample may be seriallydiluted such that only over-expressed genes remain above the detectionlimit threshold of the assay.

In certain embodiments, the present invention provides steps ofobtaining a sample of biological fluid (e.g., urine, blood plasma orserum, sputum, cerebral spinal fluid), and extracting the DNA. Themutant nucleotide sequence to be detected, may be a fraction of a largermolecule or can be present initially as a discrete molecule.

In certain embodiments, DNA is isolated from plasma/serum of a cancerpatient. For comparison, DNA samples isolated from neoplastic tissue anda second sample may be isolated from non-neoplastic tissue from the samepatient (control), for example, lymphocytes. The non-neoplastic tissuecan be of the same type as the neoplastic tissue or from a differentorgan source. In certain embodiments, blood samples are collected andplasma immediately separated from the blood cells by centrifugation.Serum may be filtered and stored frozen until DNA extraction.

In certain example embodiments, target nucleic acids are detecteddirectly from a crude or unprocessed sample, such as blood, serum,saliva, cerebrospinal fluid, sputum, or urine. In certain exampleembodiments, the target nucleic acid is cell free DNA.

Circulating Tumor Cells

In one embodiment, circulating cells (e.g., circulating tumor cells(CTC)) can be assayed with the present invention. Isolation ofcirculating tumor cells (CTC) for use in any of the methods describedherein may be performed. Exemplary technologies that achieve specificand sensitive detection and capture of circulating cells that may beused in the present invention have been described (Mostert B, et al.,Circulating tumor cells (CTCs): detection methods and their clinicalrelevance in breast cancer. Cancer Treat Rev. 2009; 35:463-474; andTalasaz A H, et al., Isolating highly enriched populations ofcirculating epithelial cells and other rare cells from blood using amagnetic sweeper device. Proc Natl Acad Sci USA. 2009; 106:3970-3975).As few as one CTC may be found in the background of 105-106 peripheralblood mononuclear cells (Ross A A, et al., Detection and viability oftumor cells in peripheral blood stem cell collections from breast cancerpatients using immunocytochemical and clonogenic assay techniques.Blood. 1993, 82:2605-2610). The Cell Search® platform usesimmunomagnetic beads coated with antibodies to Epithelial Cell AdhesionMolecule (EpCAM) to enrich for EPCAM-expressing epithelial cells,followed by immunostaining to confirm the presence of cytokeratinstaining and absence of the leukocyte marker CD45 to confirm thatcaptured cells are epithelial tumor cells (Momburg F, et al.,Immunohistochemical study of the expression of a Mr 34,000 humanepithelium-specific surface glycoprotein in normal and malignanttissues. Cancer Res. 1987; 47:2883-2891; and Allard W J, et al., Tumorcells circulate in the peripheral blood of all major carcinomas but notin healthy subjects or patients with nonmalignant diseases. Clin CancerRes. 2004; 10:6897-6904). The number of cells captured have beenprospectively demonstrated to have prognostic significance for breast,colorectal and prostate cancer patients with advanced disease (Cohen SJ, et al., J Clin Oncol. 2008; 26:3213-3221; Cristofanilli M, et al. NEngl J Med. 2004; 351:781-791; Cristofanilli M, et al., J Clin Oncol.2005; 23: 1420-1430; and de Bono J S, et al. Clin Cancer Res. 2008;14:6302-6309).

The present invention also provides for isolating CTCs with CTC-ChipTechnology. CTC-Chip is a microfluidic based CTC capture device whereblood flows through a chamber containing thousands of microposts coatedwith anti-EpCAM antibodies to which the CTCs bind (Nagrath S, et al.Isolation of rare circulating tumor cells in cancer patients bymicrochip technology. Nature. 2007; 450: 1235-1239). CTC-Chip provides asignificant increase in CTC counts and purity in comparison to theCellSearch® system (Maheswaran S, et al. Detection of mutations in EGFRin circulating lung-cancer cells, N Engl J Med. 2008; 359:366-377), bothplatforms may be used for downstream molecular analysis.

Cell-Free Chromatin

In certain embodiments, cell free chromatin fragments are isolated andanalyzed according to the present invention. Nucleosomes can be detectedin the serum of healthy individuals (Stroun et al., Annals of the NewYork Academy of Sciences 906: 161-168 (2000)) as well as individualsafflicted with a disease state. Moreover, the serum concentration ofnucleosomes is considerably higher in patients suffering from benign andmalignant diseases, such as cancer and autoimmune disease (Holdenriederet al (2001) Int J Cancer 95, 1 14-120, Trejo-Becerril et al (2003) IntJ Cancer 104, 663-668; Kuroi et al 1999 Breast Cancer 6, 361-364; Kuroiet al (2001) Int j Oncology 19, 143-148; Amoura et al (1997) Arth Rheum40, 2217-2225; Williams et al (2001) J Rheumatol 28, 81-94). Not beingbound by a theory, the high concentration of nucleosomes in tumorbearing patients derives from apoptosis, which occurs spontaneously inproliferating tumors. Nucleosomes circulating in the blood containuniquely modified histones. For example, U.S. Patent Publication No.2005/0069931 (Mar. 31, 2005) relates to the use of antibodies directedagainst specific histone N-terminus modifications as diagnosticindicators of disease, employing such histone-specific antibodies toisolate nucleosomes from a blood or serum sample of a patient tofacilitate purification and analysis of the accompanying DNA fordiagnostic/screening purposes. Accordingly, the present invention mayuse chromatin bound DNA to detect and monitor, for example, tumormutations. The identification of the DNA associated with modifiedhistones can serve as diagnostic markers of disease and congenitaldefects.

Thus, in another embodiment, isolated chromatin fragments are derivedfrom circulating chromatin, preferably circulating mono andoligonucleosomes. Isolated chromatin fragments may be derived from abiological sample. The biological sample may be from a subject or apatient in need thereof. The biological sample may be sera, plasma,lymph, blood, blood fractions, urine, synovial fluid, spinal fluid,saliva, circulating tumor cells or mucous.

Cell-Free DNA (cfDNA)

In certain embodiments, the present invention may be used to detect cellfree DNA (cfDNA). Cell free DNA in plasma or serum may be used as anon-invasive diagnostic tool. For example, cell free fetal DNA has beenstudied and optimized for testing on-compatible RhD factors, sexdetermination for X-linked genetic disorders, testing for single genedisorders, identification of preeclampsia. For example, sequencing thefetal cell fraction of cfDNA in maternal plasma is a reliable approachfor detecting copy number changes associated with fetal chromosomeaneuploidy. For another example, cfDNA isolated from cancer patients hasbeen used to detect mutations in key genes relevant for treatmentdecisions.

In certain example embodiments, the present disclosure providesdetecting cfDNA directly from a patient sample. In certain other exampleembodiment, the present disclosure provides enriching cfDNA using theenrichment embodiments disclosed above and prior to detecting the targetcfDNA.

Exosomes

In one embodiment, exosomes can be assayed with the present invention.Exosomes are small extracellular vesicles that have been shown tocontain RNA. Isolation of exosomes by ultracentrifugation, filtration,chemical precipitation, size exclusion chromatography, and microfluidicsare known in the art. In one embodiment exosomes are purified using anexosome biomarker. Isolation and purification of exosomes frombiological samples may be performed by any known methods (see e.g.,WO2016172598A1).

SNP Detection and Genotyping

In certain embodiments, the present invention may be used to detect thepresence of single nucleotide polymorphisms (SNP) in a biologicalsample. The SNPs may be related to maternity testing (e.g., sexdetermination, fetal defects). They may be related to a criminalinvestigation. In one embodiment, a suspect in a criminal investigationmay be identified by the present invention. Not being bound by a theorynucleic acid based forensic evidence may require the most sensitiveassay available to detect a suspect or victim's genetic material becausethe samples tested may be limiting.

In other embodiments, SNPs associated with a disease are encompassed bythe present invention. SNPs associated with diseases are well known inthe art and one skilled in the art can apply the methods of the presentinvention to design suitable guide RNAs (see e.g.,www.ncbi.nlm.nih.gov/clinvar?term=human%5Borgn %5D).

In an aspect, the invention relates to a method for genotyping, such asSNP genotyping, comprising:

-   -   a) distributing a sample or set of samples into one or more        individual discrete volumes, the individual discrete volumes        comprising a CRISPR system according to the invention as        described herein;    -   b) incubating the sample or set of samples under conditions        sufficient to allow binding of the one or more guide RNAs to one        or more target molecules;    -   c) activating the CRISPR effector protein via binding of the one        or more guide RNAs to the one or more target molecules, wherein        activating the CRISPR effector protein results in modification        of the RNA-based masking construct such that a detectable        positive signal is generated; and    -   d) detecting the detectable positive signal, wherein detection        of the detectable positive signal indicates a presence of one or        more target molecules characteristic for a particular genotype        in the sample.

In certain embodiments, the detectable signal is compared to (e.g. bycomparison of signal intensity) one or more standard signals, preferablya synthetic standard signal, such as for instance illustrated in anexample embodiment in FIGS. 60A-60E. In certain embodiments, thestandard is or corresponds to a particular genotype. In certainembodiments, the standard comprises a particular SNP or other (single)nucleotide variation. In certain embodiments, the standard is a(PCR-amplified) genotype standard. In certain embodiments, the standardis or comprises DNA. In certain embodiments, the standard is orcomprises RNA. In certain embodiments, the standard is or comprised RNAwhich is transcribed from DNA. In certain embodiments, the standard isor comprises DNA which is reverse transcribed from RNA. In certainembodiments, the detectable signal is compared to one or more standard,each of which corresponds to a known genotype, such as a SNP or other(single) nucleotide variation. In certain embodiments, the detectablesignal is compared to one or more standard signal and the comparisoncomprises statistical analysis, such as by parametric or non-parametricstatistical analysis, such as by one- or two-way ANOVA, etc. In certainembodiments, the detectable signal is compared to one or more standardsignal and when the detectable signal does not (statistically)significantly deviate from the standard, the genotype is determined asthe genotype corresponding to said standard.

In other embodiments, the present invention allows rapid genotyping foremergency pharmacogenomics. In one embodiment, a single point of careassay may be used to genotype a patient brought in to the emergencyroom. The patient may be suspected of having a blood clot and anemergency physician needs to decide a dosage of blood thinner toadminister. In exemplary embodiments, the present invention may provideguidance for administration of blood thinners during myocardialinfarction or stroke treatment based on genotyping of markers such asVKORC1, CYP2C9, and CYP2C19. In one embodiment, the blood thinner is theanticoagulant warfarin (Holford, N H (December 1986). “ClinicalPharmacokinetics and Pharmacodynamics of Warfarin Understanding theDose-Effect Relationship”. Clinical Pharmacokinetics. SpringerInternational Publishing. 11 (6): 483-504). Genes associated with bloodclotting are known in the art (see e.g., US20060166239A1; Litin S C,Gastineau D A (1995) “Current concepts in anticoagulant therapy”. MayoClin. Proc. 70 (3): 266-72; and Rusdiana et al., Responsiveness tolow-dose warfarin associated with genetic variants of VKORC1, CYP2C9,CYP2C19, and CYP4F2 in an Indonesian population. Eur J Clin Pharmacol.2013 March; 69(3):395-405). Specifically, in the VKORC1 1639 (or 3673)single-nucleotide polymorphism, the common (“wild-type”) G allele isreplaced by the A allele. People with an A allele (or the “A haplotype”)produce less VKORC1 than do those with the G allele (or the “non-Ahaplotype”). The prevalence of these variants also varies by race, with37% of Caucasians and 14% of Africans carrying the A allele. The endresult is a decreased number of clotting factors and therefore, adecreased ability to clot.

In certain example embodiments, the availability of genetic material fordetecting a SNP in a patient allows for detecting SNPs withoutamplification of a DNA or RNA sample. In the case of genotyping, thebiological sample tested is easily obtained. In certain exampleembodiments, the incubation time of the present invention may beshortened. The assay may be performed in a period of time required foran enzymatic reaction to occur. One skilled in the art can performbiochemical reactions in 5 minutes (e.g., 5 minute ligation). Thepresent invention may use an automated DNA extraction device to obtainDNA from blood. The DNA can then be added to a reaction that generates atarget molecule for the effector protein. Immediately upon generatingthe target molecule the masking agent can be cut and a signal detected.In exemplary embodiments, the present invention allows a POC rapiddiagnostic for determining a genotype before administering a drug (e.g.,blood thinner). In the case where an amplification step is used, all ofthe reactions occur in the same reaction in a one step process. Inpreferred embodiments, the POC assay may be performed in less than anhour, preferably 10 minutes, 20 minutes, 30 minutes, 40 minutes, or 50minutes.

In certain embodiments, the systems, devices, and methods disclosedherein may be used for detecting the presence or expression level oflong non-coding RNAs (lncRNAs). Expression of certain lncRNAs areassociated with disease state and/or drug resistance. In particular,certain lncRNAs (e.g., TCONS_00011252, NR 034078, TCONS_00010506,TCONS_00026344, TCONS_00015940, TCONS_00028298, TCONS_00026380,TCONS_0009861, TCONS_00026521, TCONS_00016127, NR 125939, NR 033834,TCONS_00021026, TCONS_00006579, NR 109890, and NR 026873) are associatedwith resistance to cancer treatment, such as resistance to one or moreBRAF inhibitors (e.g., Vemurafenib, Dabrafenib, Sorafenib, GDC-0879,PLX-4720, and LGX818) for treating melanoma (e.g., nodular melanoma,lentigo maligna, lentigo maligna melanoma, acral lentiginous melanoma,superficial spreading melanoma, mucosal melanoma, polypoid melanoma,desmoplastic melanoma, amelanotic melanoma, and soft-tissue melanoma).The detection of lncRNAs using the various embodiments described hereincan facilitate disease diagnosis and/or selection of treatment options.

In one embodiment, the present invention can guide DNA- or RNA-targetedtherapies (e.g., CRISPR, TALE, Zinc finger proteins, RNAi), particularlyin settings where rapid administration of therapy is important totreatment outcomes.

LOH Detection

Cancer cells undergo a loss of genetic material (DNA) when compared tonormal cells. This deletion of genetic material which almost all, if notall, cancers undergo is referred to as “loss of heterozygosity” (LOH).Loss of heterozygosity (LOH) is a gross chromosomal event that resultsin loss of the entire gene and the surrounding chromosomal region. Theloss of heterozygosity is a common occurrence in cancer, where it canindicate the absence of a functional tumor suppressor gene in the lostregion. However, a loss may be silent because there still is onefunctional gene left on the other chromosome of the chromosome pair. Theremaining copy of the tumor suppressor gene can be inactivated by apoint mutation, leading to loss of a tumor suppressor gene. The loss ofgenetic material from cancer cells can result in the selective loss ofone of two or more alleles of a gene vital for cell viability or cellgrowth at a particular locus on the chromosome.

An “LOH marker” is DNA from a microsatellite locus, a deletion,alteration, or amplification in which, when compared to normal cells, isassociated with cancer or other diseases. An LOH marker often isassociated with loss of a tumor suppressor gene or another, usuallytumor related, gene.

The term “microsatellites” refers to short repetitive sequences of DNAthat are widely distributed in the human genome. A microsatellite is atract of tandemly repeated (i.e. adjacent) DNA motifs that range inlength from two to five nucleotides, and are typically repeated 5-50times. For example, the sequence TATATATATA (SEQ. I.D. No. 418) is adinucleotide microsatellite, and GTCGTCGTCGTCGTC (SEQ. I.D. No. 419) isa trinucleotide microsatellite (with A being Adenine, G Guanine, CCytosine, and T Thymine). Somatic alterations in the repeat length ofsuch microsatellites have been shown to represent a characteristicfeature of tumors. Guide RNAs may be designed to detect suchmicrosatellites. Furthermore, the present invention may be used todetect alterations in repeat length, as well as amplifications anddeletions based upon quantitation of the detectable signal. Certainmicrosatellites are located in regulatory flanking or intronic regionsof genes, or directly in codons of genes. Microsatellite mutations insuch cases can lead to phenotypic changes and diseases, notably intriplet expansion diseases such as fragile X syndrome and Huntington'sdisease.

Frequent loss of heterozygosity (LOH) on specific chromosomal regionshas been reported in many kinds of malignancies. Allelic losses onspecific chromosomal regions are the most common genetic alterationsobserved in a variety of malignancies, thus microsatellite analysis hasbeen applied to detect DNA of cancer cells in specimens from bodyfluids, such as sputum for lung cancer and urine for bladder cancer.(Rouleau, et al. Nature 363, 515-521 (1993); and Latif, et al. Science260, 1317-1320 (1993)). Moreover, it has been established that markedlyincreased concentrations of soluble DNA are present in plasma ofindividuals with cancer and some other diseases, indicating that cellfree serum or plasma can be used for detecting cancer DNA withmicrosatellite abnormalities. (Kamp, et al. Science 264, 436-440 (1994);and Steck, et al. Nat Genet. 15(4), 356-362 (1997)). Two groups havereported microsatellite alterations in plasma or serum of a limitednumber of patients with small cell lung cancer or head and neck cancer.(Hahn, et al. Science 271, 350-353 (1996); and Miozzo, et al. CancerRes. 56, 2285-2288 (1996)). Detection of loss of heterozygosity intumors and serum of melanoma patients has also been previously shown(see, e.g., United States patent number U.S. Pat. No. 6,465,177B1).

Thus, it is advantageous to detect of LOH markers in a subject sufferingfrom or at risk of cancer. The present invention may be used to detectLOH in tumor cells. In one embodiment, circulating tumor cells may beused as a biological sample. In preferred embodiments, cell free DNAobtained from serum or plasma is used to noninvasively detect and/ormonitor LOH. In other embodiments, the biological sample may be anysample described herein (e.g., a urine sample for bladder cancer). Notbeing bound by a theory, the present invention may be used to detect LOHmarkers with improved sensitivity as compared to any prior method, thusproviding early detection of mutational events. In one embodiment, LOHis detected in biological fluids, wherein the presence of LOH isassociated with the occurrence of cancer. The method and systemsdescribed herein represents a significant advance over prior techniques,such as PCR or tissue biopsy by providing a non-invasive, rapid, andaccurate method for detecting LOH of specific alleles associated withcancer. Thus, the present invention provides a methods and systems whichcan be used to screen high-risk populations and to monitor high riskpatients undergoing chemoprevention, chemotherapy, immunotherapy orother treatments.

Because the method of the present invention requires only DNA extractionfrom bodily fluid such as blood, it can be performed at any time andrepeatedly on a single patient. Blood can be taken and monitored for LOHbefore or after surgery; before, during, and after treatment, such aschemotherapy, radiation therapy, gene therapy or immunotherapy; orduring follow-up examination after treatment for disease progression,stability, or recurrence. Not being bound by a theory, the method of thepresent invention also may be used to detect subclinical diseasepresence or recurrence with an LOH marker specific for that patientsince LOH markers are specific to an individual patient's tumor. Themethod also can detect if multiple metastases may be present using tumorspecific LOH markers.

Detection of Epigenetic Modifications

Histone variants, DNA modifications, and histone modificationsindicative of cancer or cancer progression may be used in the presentinvention. For example, U.S. patent publication 20140206014 describesthat cancer samples had elevated nucleosome H2AZ, macroH2A1.1,5-methylcytosine, P-H2AX(Ser139) levels as compared to healthy subjects.The presence of cancer cells in an individual may generate a higherlevel of cell free nucleosomes in the blood as a result of the increasedapoptosis of the cancer cells. In one embodiment, an antibody directedagainst marks associated with apoptosis, such as H2B Ser 14(P), may beused to identify single nucleosomes that have been released fromapoptotic neoplastic cells. Thus, DNA arising from tumor cells may beadvantageously analyzed according to the present invention with highsensitivity and accuracy.

Pre-Natal Screening

In certain embodiments, the method and systems of the present inventionmay be used in prenatal screening. In certain embodiments, cell-free DNAis used in a method of prenatal screening. In certain embodiments, DNAassociated with single nucleosomes or oligonucleosomes may be detectedwith the present invention. In preferred embodiments, detection of DNAassociated with single nucleosomes or oligonucleosomes is used forprenatal screening. In certain embodiments, cell-free chromatinfragments are used in a method of prenatal screening.

Prenatal diagnosis or prenatal screening refers to testing for diseasesor conditions in a fetus or embryo before it is born. The aim is todetect birth defects such as neural tube defects, Down syndrome,chromosome abnormalities, genetic disorders and other conditions, suchas spina bifida, cleft palate, Tay Sachs disease, sickle cell anemia,thalassemia, cystic fibrosis, Muscular dystrophy, and fragile Xsyndrome. Screening can also be used for prenatal sex discernment.Common testing procedures include amniocentesis, ultrasonographyincluding nuchal translucency ultrasound, serum marker testing, orgenetic screening. In some cases, the tests are administered todetermine if the fetus will be aborted, though physicians and patientsalso find it useful to diagnose high-risk pregnancies early so thatdelivery can be scheduled in a tertian,′ care hospital where the babycan receive appropriate care.

It has been realized that there are fetal cells which are present in themother's blood, and that these cells present a potential source of fetalchromosomes for prenatal DNA-based diagnostics. Additionally, fetal DNAranges from about 2-10% of the total DNA in maternal blood. Currentlyavailable prenatal genetic tests usually involve invasive procedures.For example, chorionic villus sampling (CVS) performed on a pregnantwoman around 10-12 weeks into the pregnancy and amniocentesis performedat around 14-16 weeks all contain invasive procedures to obtain thesample for testing chromosomal abnormalities in a fetus. Fetal cellsobtained via these sampling procedures are usually tested forchromosomal abnormalities using cytogenetic or fluorescent in situhybridization (FISH) analyses. Cell-free fetal DNA has been shown toexist in plasma and serum of pregnant women as early as the sixth weekof gestation, with concentrations rising during pregnancy and peakingprior to parturition. Because these cells appear very early in thepregnancy, they could form the basis of an accurate, noninvasive, firsttrimester test. Not being bound by a theory, the present inventionprovides unprecedented sensitivity in detecting low amounts of fetalDNA. Not being bound by a theory, abundant amounts of maternal DNA isgenerally concomitantly recovered along with the fetal DNA of interest,thus decreasing sensitivity in fetal DNA quantification and mutationdetection. The present invention overcomes such problems by theunexpectedly high sensitivity of the assay.

The H3 class of histones consists of four different protein types: themain types, H3.1 and H3.2; the replacement type, H3.3; and the testisspecific variant, H3t. Although H3.1 and H3.2 are closely related, onlydiffering at Ser96, H3.1 differs from H3.3 in at least 5 amino acidpositions. Further, H3.1 is highly enriched in fetal liver, incomparison to its presence in adult tissues including liver, kidney andheart. In adult human tissue, the H3.3 variant is more abundant than theH3.1 variant, whereas the converse is true for fetal liver. The presentinvention may use these differences to detect fetal nucleosomes andfetal nucleic acid in a maternal biological sample that comprises bothfetal and maternal cells and/or fetal nucleic acid.

In one embodiment, fetal nucleosomes may be obtained from blood. Inother embodiments, fetal nucleosomes are obtained from a cervical mucussample. In certain embodiments, a cervical mucus sample is obtained byswabbing or lavage from a pregnant woman early in the second trimesteror late in the first trimester of pregnancy. The sample may be placed inan incubator to release DNA trapped in mucus. The incubator may be setat 37° C. The sample may be rocked for approximately 15 to 30 minutes.Mucus may be further dissolved with a mucinase for the purpose ofreleasing DNA. The sample may also be subjected to conditions, such aschemical treatment and the like, as well known in the art, to induceapoptosis to release fetal nucleosomes. Thus, a cervical mucus samplemay be treated with an agent that induces apoptosis, whereby fetalnucleosomes are released. Regarding enrichment of circulating fetal DNA,reference is made to U.S. patent publication Nos. 20070243549 and20100240054. The present invention is especially advantageous whenapplying the methods and systems to prenatal screening where only asmall fraction of nucleosomes or DNA may be fetal in origin.

Prenatal screening according to the present invention may be for adisease including, but not limited to Trisomy 13, Trisomy 16, Trisomy18, Klinefelter syndrome (47, XXY), (47, XYY) and (47, XXX), Turnersyndrome, Down syndrome (Trisomy 21), Cystic Fibrosis, Huntington'sDisease, Beta Thalassaemia, Myotonic Dystrophy, Sickle Cell Anemia,Porphyria, Fragile-X-Syndrome, Robertsonian translocation, Angelmansyndrome, DiGeorge syndrome and Wolf-Hirschhorn Syndrome.

Several further aspects of the invention relate to diagnosing,prognosing and/or treating defects associated with a wide range ofgenetic diseases which are further described on the website of theNational Institutes of Health under the topic subsection GeneticDisorders (website at health.nih.gov/topic/Genetic Disorders).

Cancer and Cancer Drug Resistance Detection

In certain embodiments, the present invention may be used to detectgenes and mutations associated with cancer. In certain embodiments,mutations associated with resistance are detected. The amplification ofresistant tumor cells or appearance of resistant mutations in clonalpopulations of tumor cells may arise during treatment (see, e.g., BurgerJ A, et al., Clonal evolution in patients with chronic lymphocyticleukaemia developing resistance to BTK inhibition. Nat Commun. 2016 May20; 7:11589; Landau D A, et al., Mutations driving CLL and theirevolution in progression and relapse. Nature. 2015 Oct. 22;526(7574):525-30; Landau D A, et al., Clonal evolution in hematologicalmalignancies and therapeutic implications. Leukemia. 2014 January;28(1):34-43; and Landau D A, et al., Evolution and impact of subclonalmutations in chronic lymphocytic leukemia. Cell. 2013 Feb. 14;152(4):714-26). Accordingly, detecting such mutations requires highlysensitive assays and monitoring requires repeated biopsy. Repeatedbiopsies are inconvenient, invasive and costly. Resistant mutations canbe difficult to detect in a blood sample or other noninvasivelycollected biological sample (e.g., blood, saliva, urine) using the priormethods known in the art. Resistant mutations may refer to mutationsassociated with resistance to a chemotherapy, targeted therapy, orimmunotherapy.

In certain embodiments, mutations occur in individual cancers that maybe used to detect cancer progression. In one embodiment, mutationsrelated to T cell cytolytic activity against tumors have beencharacterized and may be detected by the present invention (see e.g.,Rooney et al., Molecular and genetic properties of tumors associatedwith local immune cytolytic activity, Cell. 2015 January 15; 160(1-2):48-61). Personalized therapies may be developed for a patient based ondetection of these mutations (see e.g., WO2016100975A1). In certainembodiments, cancer specific mutations associated with cytolyticactivity may be a mutation in a gene selected from the group consistingof CASP8, B2M, PIK3CA, SMC1A, ARID5B, TET2, ALPK2, COL5A1, TP53, DNER,NCOR1, MORC4, CIC, IRF6, MYOCD, ANKLE1, CNKSR1, NF1, SOS1, ARID2, CUL4B,DDX3X, FUBP1, TCP11L2, HLA-A, B or C, CSNK2A1, MET, ASXL1, PD-L1, PD-L2,IDO1, IDO2, ALOX12B and ALOX15B, or copy number gain, excludingwhole-chromosome events, impacting any of the following chromosomalbands: 6q16.1-q21, 6q22.31-q24.1, 6q25.1-q26, 7p11.2-q11.1, 8p23.1,8p11.23-p11.21 (containing IDO1, IDO2), 9p24.2-p23 (containing PDL1,PDL2), 10p15.3, 10p15.1-p13, 11p14.1, 12p13.32-p13.2, 17p13.1(containing ALOX12B, ALOX15B), and 22q11.1-q11.21.

In certain embodiments, the present invention is used to detect a cancermutation (e.g., resistance mutation) during the course of a treatmentand after treatment is completed. The sensitivity of the presentinvention may allow for noninvasive detection of clonal mutationsarising during treatment and can be used to detect a recurrence in thedisease.

In certain example embodiments, detection of microRNAs (miRNA) and/ormiRNA signatures of differentially expressed miRNA, may be used todetect or monitor progression of a cancer and/or detect drug resistanceto a cancer therapy. As an example, Nadal et al. (Nature ScientificReports, (2015) doi:10.1038/srep12464) describe mRNA signatures that maybe used to detect non-small cell lung cancer (NSCLC).

In certain example embodiments, the presence of resistance mutations inclonal subpopulations of cells may be used in determining a treatmentregimen. In other embodiments, personalized therapies for treating apatient may be administered based on common tumor mutations. In certainembodiments, common mutations arise in response to treatment and lead todrug resistance. In certain embodiments, the present invention may beused in monitoring patients for cells acquiring a mutation oramplification of cells harboring such drug resistant mutations.

Treatment with various chemotherapeutic agents, particularly withtargeted therapies such as tyrosine kinase inhibitors, frequently leadsto new mutations in the target molecules that resist the activity of thetherapeutic. Multiple strategies to overcome this resistance are beingevaluated, including development of second generation therapies that arenot affected by these mutations and treatment with multiple agentsincluding those that act downstream of the resistance mutation. In anexemplary embodiment, a common mutation to ibrutinib, a moleculetargeting Bruton's Tyrosine Kinase (BTK) and used for CLL and certainlymphomas, is a Cysteine to Serine change at position 481 (BTK/C481S).Erlotinib, which targets the tyrosine kinase domain of the EpidermalGrowth Factor Receptor (EGFR), is commonly used in the treatment of lungcancer and resistant tumors invariably develop following therapy. Acommon mutation found in resistant clones is a threonine to methioninemutation at position 790.

Non-silent mutations shared between populations of cancer patients andcommon resistant mutations that may be detected with the presentinvention are known in the art (see e.g., WO/2016/187508). In certainembodiments, drug resistance mutations may be induced by treatment withibrutinib, erlotinib, imatinib, gefitinib, crizotinib, trastuzumab,vemurafenib, RAF/MEK, check point blockade therapy, or antiestrogentherapy. In certain embodiments, the cancer specific mutations arepresent in one or more genes encoding a protein selected from the groupconsisting of Programmed Death-Ligand 1 (PD-L1), androgen receptor (AR),Bruton's Tyrosine Kinase (BTK), Epidermal Growth Factor Receptor (EGFR),BCR-Abl, c-kit, PIK3CA, HER2, EML4-ALK, KRAS, ALK, ROS1, AKT1, BRAF,MEK1, MEK2, NRAS, RAC1, and ESR1.

Immune checkpoints are inhibitory pathways that slow down or stop immunereactions and prevent excessive tissue damage from uncontrolled activityof immune cells. In certain embodiments, the immune checkpoint targetedis the programmed death-1 (PD-1 or CD279) gene (PDCD1). In otherembodiments, the immune checkpoint targeted is cytotoxicT-lymphocyte-associated antigen (CTLA-4). In additional embodiments, theimmune checkpoint targeted is another member of the CD28 and CTLA4 Igsuperfamily such as BTLA, LAG3, ICOS, PDL1 or KIR. In further additionalembodiments, the immune checkpoint targeted is a member of the TNFRsuperfamily such as CD40, OX40, CD137, GITR, CD27 or TIM-3.

Recently, gene expression in tumors and their microenvironments havebeen characterized at the single cell level (see e.g., Tirosh, et al.Dissecting the multicellular ecosystem of metastatic melanoma by singlecell RNA-seq. Science 352, 189-196, doi:10.1126/science.aad0501 (2016));Tirosh et al., Single-cell RNA-seq supports a developmental hierarchy inhuman oligodendroglioma. Nature. 2016 Nov. 10; 539(7628):309-313. doi:10.1038/nature20123. Epub 2016 Nov. 2; and International patentpublication serial number WO 2017004153 A1). In certain embodiments,gene signatures may be detected using the present invention. In oneembodiment complement genes are monitored or detected in a tumormicroenvironment. In one embodiment MITF and AXL programs are monitoredor detected. In one embodiment, a tumor specific stem cell or progenitorcell signature is detected. Such signatures indicate the state of animmune response and state of a tumor. In certain embodiments, the stateof a tumor in terms of proliferation, resistance to treatment andabundance of immune cells may be detected.

Thus, in certain embodiments, the invention provides low-cost, rapid,multiplexed cancer detection panels for circulating DNA, such as tumorDNA, particularly for monitoring disease recurrence or the developmentof common resistance mutations.

Immunotherapy Applications

The embodiments disclosed herein can also be useful in furtherimmunotherapy contexts. For instance, in some embodiments methods ofdiagnosing, prognosing and/or staging an immune response in a subjectcomprise detecting a first level of expression, activity and/or functionof one or more biomarker and comparing the detected level to a controllevel wherein a difference in the detected level and the control levelindicates that the presence of an immune response in the subject.

In certain embodiments, the present invention may be used to determinedysfunction or activation of tumor infiltrating lymphocytes (TIL). TILsmay be isolated from a tumor using known methods. The TILs may beanalyzed to determine whether they should be used in adoptive celltransfer therapies. Additionally, chimeric antigen receptor T cells (CART cells) may be analyzed for a signature of dysfunction or activationbefore administering them to a subject. Exemplary signatures fordysfunctional and activated T cell have been described (see e.g., SingerM, et al., A Distinct Gene Module for Dysfunction Uncoupled fromActivation in Tumor-Infiltrating T Cells. Cell. 2016 Sep. 8;166(6):1500-1511.e9. doi: 10.1016/j.cell.2016.08.052).

In some embodiments, C2c2 is used to evaluate that state of immunecells, such as T cells (e.g., CD8+ and/or CD4+ T cells). In particular,T cell activation and/or dysfunction can be determined, e.g., based ongenes or gene signatures associated with one or more of the T cellstates. In this way, c2c2 can be used to determine the presence of oneor more subpopulations of T cells.

In some embodiments, C2c2 can be used in a diagnostic assay or may beused as a method of determining whether a patient is suitable foradministering an immunotherapy or another type of therapy. For example,detection of gene or biomarker signatures may be performed via c2c2 todetermine whether a patient is responding to a given treatment or, ifthe patient is not responding, if this may be due to T cell dysfunction.Such detection is informative regarding the types of therapy the patientis best suited to receive. For example, whether the patient shouldreceive immunotherapy.

In some embodiments, the systems and assays disclosed herein may allowclinicians to identify whether a patient's response to a therapy (e.g.,an adoptive cell transfer (ACT) therapy) is due to cell dysfunction, andif it is, levels of up-regulation and down-regulation across thebiomarker signature will allow problems to be addressed. For example, ifa patient receiving ACT is non-responsive, the cells administered aspart of the ACT may be assayed by an assay disclosed herein to determinethe relative level of expression of a biomarker signature known to beassociated with cell activation and/or dysfunction states. If aparticular inhibitory receptor or molecule is up-regulated in the ACTcells, the patient may be treated with an inhibitor of that receptor ormolecule. If a particular stimulatory receptor or molecule isdown-regulated in the ACT cells, the patient may be treated with anagonist of that receptor or molecule.

In certain example embodiments, the systems, methods, and devicesdescribed herein may be used to screen gene signatures that identify aparticular cell type, cell phenotype, or cell state. Likewise, throughthe use of such methods as compressed sensing, the embodiments disclosedherein may be used to detect transcriptomes. Gene expression data arehighly structured, such that the expression level of some genes ispredictive of the expression level of others. Knowledge that geneexpression data are highly structured allows for the assumption that thenumber of degrees of freedom in the system are small, which allows forassuming that the basis for computation of the relative gene abundancesis sparse. It is possible to make several biologically motivatedassumptions that allow Applicants to recover the nonlinear interactionterms while under-sampling without having any specific knowledge ofwhich genes are likely to interact. In particular, if Applicants assumethat genetic interactions are low rank, sparse, or a combination ofthese, then the true number of degrees of freedom is small relative tothe complete combinatorial expansion, which enables Applicants to inferthe full nonlinear landscape with a relatively small number ofperturbations. Working around these assumptions, analytical theories ofmatrix completion and compressed sensing may be used to designunder-sampled combinatorial perturbation experiments. In addition, akernel-learning framework may be used to employ under-sampling bybuilding predictive functions of combinatorial perturbations withoutdirectly learning any individual interaction coefficient Compressessensing provides a way to identify the minimal number of targettranscripts to be detected in order obtain a comprehensivegene-expression profile. Methods for compressed sensing are disclosed inPCT/US2016/059230 “Systems and Methods for Determining RelativeAbundances of Biomolecules” filed Oct. 27, 2016, which is incorporatedherein by reference. Having used methods like compressed sensing toidentify a minimal transcript target set, a set of corresponding guideRNAs may then be designed to detect said transcripts.

Accordingly, in certain example embodiments, a method for obtaining agene-expression profile of cell comprises detecting, using theembodiments disclosed, herein a minimal transcript set that provides agene-expression profile of a cell or population of cells.

Detecting Gene Edits and/or Off-Target Effects

The embodiments disclosed herein may be used in combination with othergene editing tools to confirm that a desired genetic edit or edits weresuccessful and/or to detect the presence of any off-target effects.Cells that have been edited may be screened using one or more guides toone or more target loci. As the embodiments disclosed herein utilizeCRISPR systems, theranostic applications are also envisioned. Forexample, genotyping embodiments disclosed herein may be used to selectappropriate target loci or identify cells or populations of cells inneeded of the target edit. The same or separate system may then be usedto determine editing efficiency. As described in the Working Examplesbelow, the embodiments disclosed herein may be used to designstreamlined theranostic pipelines in as little as one week.

Detecting Nucleic Acid Tagged Items

Alternatively, the embodiments described herein may be used to detectnucleic acid identifiers. Nucleic acid identifiers are non-codingnucleic acids that may be used to identify a particular article. Examplenucleic acid identifiers, such as DNA watermarks, are described inHeider and Barnekow. “DNA watermarks: A proof of concept” BMC MolecularBiology 9:40 (2008). The nucleic acid identifiers may also be a nucleicacid barcode. A nucleic-acid based barcode is a short sequence ofnucleotides (for example, DNA, RNA, or combinations thereof) that isused as an identifier for an associated molecule, such as a targetmolecule and/or target nucleic acid. A nucleic acid barcode can have alength of at least, for example, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14,15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 40,45, 50, 60, 70, 80, 90, or 100 nucleotides, and can be in single- ordouble-stranded form. One or more nucleic acid barcodes can be attached,or “tagged,” to a target molecule and/or target nucleic acid. Thisattachment can be direct (for example, covalent or non-covalent bindingof the barcode to the target molecule) or indirect (for example, via anadditional molecule, for example, a specific binding agent, such as anantibody (or other protein) or a barcode receiving adaptor (or othernucleic acid molecule). Target molecule and/or target nucleic acids canbe labeled with multiple nucleic acid barcodes in combinatorial fashion,such as a nucleic acid barcode concatemer. Typically, a nucleic acidbarcode is used to identify target molecules and/or target nucleic acidsas being from a particular compartment (for example a discrete volume),having a particular physical property (for example, affinity, length,sequence, etc.), or having been subject to certain treatment conditions.Target molecule and/or target nucleic acid can be associated withmultiple nucleic acid barcodes to provide information about all of thesefeatures (and more). Methods of generating nucleic acid-barcodes aredisclosed, for example, in International Patent Application PublicationNo. WO/2014/047561.

Enzymes

The application further provides orthologs of C2c2 which demonstraterobust activity making them particularly suitable for differentapplications of RNA cleavage and detection. These applications includebut are not limited to those described herein. More particularly, anortholog which is demonstrated to have stronger activity than otherstested is the C2c2 ortholog identified from the organism Leptotrichiawadei (LwC2c2). The application thus provides methods for modifying atarget locus of interest, comprising delivering to said locus anon-naturally occurring or engineered composition comprising a C2c2effector protein, more particularly a C2c2 effector protein withincreased activity as described herein and one or more nucleic acidcomponents, wherein at least the one or more nucleic acid components isengineered, the one or more nucleic acid components directs the complexto the target of interest and the effector protein forms a complex withthe one or more nucleic acid components and the complex binds to thetarget locus of interest. In particular embodiments, the target locus ofinterest comprises RNA. The application further provides for the use ofthe Cc2 effector proteins with increased activity in RNA sequencespecific interference, RNA sequence specific gene regulation, screeningof RNA or RNA products or lincRNA or non-coding RNA, or nuclear RNA, ormRNA, mutagenesis, Fluorescence in situ hybridization, or breeding.

The invention is further described in the following examples, which donot limit the scope of the invention described in the claims.

WORKING EXAMPLES Example 1—General Protocols

Provided are two ways to perform a C2c2 diagnostic test for DNA and RNA.This protocol may also be used with protein detection variants afterdelivery of the detection aptamers. The first is a two step reactionwhere amplification and C2c2 detection are done separately. The secondis where everything is combined in one reaction and this is called atwo-step reaction. It is important to keep in mind that amplificationmight not be necessary for higher concentration samples so it's good tohave a separate C2c2 protocol that doesn't have amplification built in.

CRISPR Effector Only—No Amplification:

Component Volume (μL) Protein (44 nM final) 2 crRNA (12 nM final) 1background target (100 ng total) 1 Target RNA (variable) 1 RNA sensorprobe (125 nM) 4 MgCl₂ (6 mM final) 2 Reaction Buffer 10× 2 RNAseInhibitors (murine from NEB) 2 H₂O 5 total 20

Reaction buffer is: 40 mM Tris-HCl, 60 mM NaCl, pH 7.3

Perform this reaction for 20 min-3 hrs at 37° C. Read out withexcitation: 485 nm/20 nm, emission: 528 nm/20 nm. A signal for singlemolecule sensitivity may be detected beginning at 20 min but of coursesensitivity is higher for longer reaction times.

Two Step Reaction: RPA Amplification Mix

Component Volume (μL) Primer A (100 μM) 0.48 Primer B (100 μM) 0.48 RPABuffer 59 MgAc 5 Target (variable concentration) 5 ATP (100 μM from NEBkit) 2 GTP (100 μM from NEB kit) 2 UTP (100 μM from NEB kit) 2 CTP (100μM from NEB kit) 2 T7 Polymerase (from NEB kit) 2 H₂O 25 total 104.96

Mix this reaction together and then re-suspend two to three tubes offreeze-dried enzyme mix). Add 5 μL of 280 mM MgAc to the mix to beginthe reaction. Preform reaction for 10-20 min. Each reaction is 20 μL sothis is enough for up to five reactions.

C2c2 Detection Mix

Volume Component (μL) Protein (44 nM final) 2 crRNA (12 nM final) 1background target (100 ng total) 1 RPA reaction 1 RNA sensor probe (125nM) 4 MgCl₂ (6 mM final) 2 Reaction Buffer 10× 2 RNAse Inhibitors(murine from NEB) 2 H₂O 5 total 20

Reaction buffer is: 40 mM Tris-HCl, 60 mM NaCl, pH 7.3

Perform this for 20 min-3 hours. Minimum detection time is about 20 minto see single molecule sensitivity. Performing the reaction for longeronly boosts the sensitivity.

One Pot Reaction:

Component Volume (μL) Primer A (100 μM) 0.48 Primer B (100 μM) 0.48 RPABuffer 59 MgAc 5 Lw2C2c2 (44 nM final) 2 crRNA (12 nM final) 2Background RNA (from 250 ng/μL) 2 RNAse alert substr (after resuspendingin 20 μL) 5 murine RNAse inhib from NEB 10 Target (variableconcentration) 5 ATP (100 μM from NEB kit) 2 GTP (100 μM from NEB kit) 2UTP (100 μM from NEB kit) 2 CTP (100 μM from NEB kit) 2 T7 Polymerase(from NEB kit) 2 H₂O 4 total 104.96

The NEB kit referenced is the HighScribe T7 High Yield Kit. To resuspendbuffer, use a 1.5× concentration: resuspend three tubes of freeze driedsubstrate in 59 μL of buffer and use in the mix above. Each reaction is20 μL so this is enough for 5 reactions worth. Single moleculesensitivity with this reaction has been observed in as early as 30-40min.

Example 2—C2c2 from Leptotrichia wadei Mediates Highly Sensitive andSpecific Detection of DNA and RNA

Rapid, inexpensive, and sensitive nucleic acid detection may aidpoint-of-care pathogen detection, genotyping, and disease monitoring.The RNA-guided, RNA-targeting CRISPR effector Cas13a (previously knownas C2c2) exhibits a “collateral effect” of promiscuous RNAse activityupon target recognition. Applicant combined the collateral effect ofCas13a with isothermal amplification to establish a CRISPR-baseddiagnostic (CRISPR-Dx), providing rapid DNA or RNA detection withattomolar sensitivity and single-base mismatch specificity. Applicantused this Cas13a-based molecular detection platform, termed SHERLOCK(Specific High Sensitivity Enzymatic Reporter UnLOCKing), to detectspecific strains of Zika and Dengue virus, distinguish pathogenicbacteria, genotype human DNA, and identify cell-free tumor DNAmutations. Furthermore, SHERLOCK reaction reagents can be lyophilizedfor cold-chain independence and long-term storage, and readilyreconstituted on paper for field applications.

The ability to rapidly detect nucleic acids with high sensitivity andsingle-base specificity on a portable platform may aid in diseasediagnosis and monitoring, epidemiology, and general laboratory tasks.Although methods exist for detecting nucleic acids (1-6), they havetrade-offs among sensitivity, specificity, simplicity, cost, and speed.Microbial Clustered Regularly Interspaced Short Palindromic Repeats(CRISPR) and CRISPR-associated (CRISPR-Cas) adaptive immune systemscontain programmable endonucleases that can be leveraged forCRISPR-based diagnostics (CRISPR-Dx). While some Cas enzymes target DNA(7, 8), single effector RNA-guided RNases, such as Cas13a (previouslyknown as C2c2) (8), can be reprogrammed with CRISPR RNAs (crRNAs) (9-11)to provide a platform for specific RNA sensing. Upon recognition of itsRNA target, activated Cas13a engages in “collateral” cleavage of nearbynon-targeted RNAs (10). This crRNA-programmed collateral cleavageactivity allows Cas13a to detect the presence of a specific RNA in vivoby triggering programmed cell death (10) or in vitro by nonspecificdegradation of labeled RNA (10, 12). Here Applicant describes SHERLOCK(Specific High Sensitivity Enzymatic Reporter UnLOCKing), an in vitronucleic acid detection platform with attomolar sensitivity based onnucleic acid amplification and 3 Cas13a-mediated collateral cleavage ofa commercial reporter RNA (12), allowing for real-time detection of thetarget (FIG. 17).

Methods Cloning of C2c2 Loci and Proteins for Expression

For the bacterial in vivo efficiency assay, C2c2 proteins fromLeptotrichia wadei F0279 and Leptotrichia shahii were ordered ascodon-optimized genes for mammalian expression (Genscript, Jiangsu,China) and cloned into pACYC184 backbones along with the correspondingdirect repeats flanking either a beta-lactamase targeting ornon-targeting spacer. Spacer expression was driven by a J23119 promoter.

For protein purification, mammalian codon-optimized C2c2 proteins werecloned into bacterial expression vector for protein purification(6×His/Twin Strep SUMO, a pET-based expression vector received as a giftfrom Ilya Finkelstein).

Bacterial In Vivo C2c2 Efficiency Assay

LwC2c2 and LshC2c2 in vivo efficiency plasmids and a previouslydescribed beta-lactamase plasmid (Abudayyeh 2016) were co-transformedinto NovaBlue Singles competent cells (Millipore) at 90 ng and 25 ng,respectively. After transformation, dilutions of cells were plated onampicillin and choramphicol LB-agar plate and incubated overnight at 37C. Colonies were counted the next day.

Nucleic acid target and crRNA preparation

Nucleic acid targets were PCR amplified with KAPA Hifi Hot Start (KapaBiosystems), gel extracted and purified using MinElute gel extractionkit (Qiagen). Purified dsDNA was incubated with T7 polymerase overnightat 30° C. using the HiScribe T7 Quick High Yield RNA Synthesis kit (NewEngland Biolabs) and RNA was purified with the MEGAclear TranscriptionClean-up kit (Thermo Fisher).

For preparation of crRNA, constructs were ordered as DNA (Integrated DNATechnologies) with an appended T7 promoter sequence. crRNA DNA wasannealed to a short T7 primer (final concentrations 10 uM) and incubatedwith T7 polymerase overnight at 37° C. using the HiScribe T7 Quick HighYield RNA Synthesis kit (New England Biolabs). crRNA were purified usingRNAXP clean beads (Beckman Coulter) at 2× ratio of beads to reactionvolume, with an additional 1.8× supplementation of isopropanol (Sigma).

NASBA Isothermal Amplification

Details of NASBA reaction are described in [Pardee 2016]. For a 20 μLtotal reaction volume, 6.7 μL of reaction buffer (Life Sciences,NECB-24), 3.3 μL of Nucleotide Mix (Life Sciences, NECN-24), 0.5 μL ofnuclease-free water, 0.4 μL of 12.5 μM NASBA primers, 0.1 uL of RNaseinhibitor (Roche, 03335402001) and 4 μL of RNA amplicon (or water forthe negative control) were assembled at 4° C. and incubated 65° C. for 2min and then 41° C. for 10 min. 5 μL of enzyme mix (Life Sciences,NEC-1-24) was added to each reaction, and the reaction mixture wasincubated at 41° C. for 2 hr. NASBA primers used were5′-AATTCTAATACGACTCACTATAGGGGGATCCTCTAGAAATATGGATT-3′ (SEQ ID NO. 16)and 5′-CTCGTATGTTGTGTGGAATTGT-3′ (SEQ ID NO. 17), and the underlinedpart indicates T7 promoter sequence.

Recombinase Polymerase Amplification

Primers for RPA were designed using NCBI Primer blast (Ye et al., BMCBioinformaics 13, 134 (2012) using default parameters, with theexception of amplicon size (between 100 and 140 nt), primer meltingtemperatures (between 54C and 67C) and primer size (between 30 and 35nt). Primers were then ordered as DNA (Integrated DNA Technologies).

RPA and RT-RPA reactions run were as instructed with TwistAmp® Basic orTwistAmp® Basic RT (TwistDx), respectively, with the exception that 280mM MgAc was added prior to the input template. Reactions were run with 1uL of input for 2 hr at 37 C, unless otherwise described.

LwC2c2 Protein Purification

C2c2 bacterial expression vectors were transformed into Rosetta™ 2(DE3)pLysS Singles Competent Cells (Millipore). A 16 mL starter culture wasgrown in Terrific Broth 4 growth media (12 g/L tryptone, 24 g/L yeastextract, 9.4 g/L K2HPO, 2.2 g/L KH2PO4, Sigma) (TB) was used toinoculate 4 L of TB, which was incubated at 37 C, 300 RPM until an OD600of 0.6. At this time, protein expression was induced by supplementationwith IPTG (Sigma) to a final concentration of 500 uM, and cells werecooled to 18 C for 16 h for protein expression. Cells were thencentrifuged at 5200 g, 15 min, 4 C. Cell pellet was harvested and storedat −80C for later purification.

All subsequent steps of the protein purification are performed at 4 C.Cell pellet was crushed and resuspended in lysis buffer (20 mM Tris-Hcl,500 mM NaCl, 1 mM DTT, pH 8.0) supplemented with protease inhibitors(Complete Ultra EDTA-free tablets), lysozyme, and benzonase followed bysonication (Sonifier 450, Branson, Danbury, Conn.) with the followingconditions: amplitude of 100 for 1 second on and 2 seconds off with atotal sonication time of 10 minutes. Lysate was cleared bycentrifugation for 1 hour at 4C at 10,000 g and the supernatant wasfiltered through a Stericup 0.22 micron filter (EMD Millipore). Filteredsupernatant was applied to StrepTactin Sepharose (GE) and incubated withrotation for 1 hour followed by washing of the protein-bound StrepTactinresin three times in lysis buffer. The resin was resuspended in SUMOdigest buffer (30 mM Tris-HCl, 500 mM NaCl 1 mM DTT, 0.15% Igepal(NP-40), pH 8.0) along with 250 Units of SUMO protease (ThermoFisher)and incubated overnight at 4C with rotation. Digestion was confirmed bySDS-PAGE and Commassie Blue staining and the protein eluate was isolatedby spinning the resin down. Protein was loaded onto a 5 mL HiTrap SP HPcation exchange column (GE Healthcare Life Sciences) via FPLC (AKTAPURE, GE Healthcare Life Sciences) and eluted over a salt gradient from130 mM to 2M NaCl in elution buffer (20 mM Tris-HCl, 1 mM DTT, 5%Glycerol, pH 8.0). The resulting fractions were tested for presence ofLwC2c2 by SDS-PAGE and fractions containing the protein were pooled andconcentrated via a Centrifugal Filter Unit to 1 mL in S200 buffer (10 mMHEPES, 1M NaCl, 5 mM MgCl2, 2 mM DTT, pH 7.0). The concentrated proteinwas loaded onto a gel filtration column (Superdex® 200 Increase 10/300GL, GE Healthcare Life Sciences) via FPLC. The resulting fractions fromgel filtration were analyzed by SDS-PAGE and fractions containing LwC2c2were pooled and buffer exchanged into Storage Buffer (600 mM NaCl, 50 mMTris-HCl pH 7.5, 5% Glycerol, 2 mM DTT) and frozen at −80C for storage.

LwC2c2 Collateral Detection

Detection assays were performed with 45 nM purified LwC2c2, 22.5 nMcrRNA, 125 nM substrate reporter (Thermo Scientific RNAse Alert v2), 2μL murine RNase inhibitors, 100 ng of background total RNA and varyingamounts of input nucleic acid target, unless otherwise indicated, innuclease assay buffer (40 mM Tris-HCl, 60 mM NaCl, 6 mM MgCl2, pH 7.3).If the input was amplified DNA including a T7 promoter from a RPAreaction, the above C2c2 reaction was modified to include 1 mM ATP, 1 mMGTP, 1 mM UTP, 1 mM CTP and 0.6 μL T7 polymerase mix (NEB). Reactionswere allowed to proceed for 1-3 hours at 37° C. (unless otherwiseindicated) on a fluorescent plate reader (BioTek) with fluorescentkinetics measured every 5 minutes.

The one-pot reaction combining, RPA-DNA amplification, T7 polymeraseconversion of DNA to RNA and C2c2 detection was performed by integratingthe reaction conditions above with the RPA amplification mix. Briefly,in a 50 μL one-pot assay consisted of 0.48 μM forward primer, 0.48 μMreverse primer, 1×RPA rehydration buffer, varying amounts of DNA input,45 nM LwC2c2 recombinant protein, 22.5 nM crRNA, 250 ng background totalRNA, 200 nM substrate reporter (RNase alert v2), 4 uL RNase inhibitor, 2mM ATP, 2 mM GTP, 2 mM UTP, 2 mM CTP, 1 μL T7 polymerase mix, 5 mMMgCl2, and 14 mM MgAc.

Quantitative PCR (qPCR) Analysis with TaqMan Probes

To compare SHERLOCK quantification with other established methods, qPCRon a dilution series of ssDNA 1 was performed. A TaqMan probe and primerset (sequences below) were designed against ssDNA 1 and synthesized withIDT. Assays were performed using the TaqMan Fast Advanced Master Mix(Thermo Fisher) and measured on a Roche LightCycler 480.

Table of qPCR primer/probe sequences. Name Sequence Forward PrimerGTG GAA TTG TGA GCG GAT AAA C (SEQ ID NO: 420) Reverse PrimerAAC AGC AAT CTA CTC GAC CTG (SEQ ID NO: 421) TaqMan Probe/56-FAM/AGGAAACAG/ZEN/ CTATGACCATGATTACGCC/3IABkFQ/(SEQ ID NOs: 422 and 423)Real-Time RPA with SYBR Green II

To compare SHERLOCK quantification with other established methods,Applicant performed RPA on a dilution series of ssDNA 1. To quantitateaccumulation of DNA in real-time, Applicant added 1×SYBR Green II(Thermo Fisher) to the typical RPA reaction mixture described above,which provides a fluorescent signal that correlates with the amount ofnucleic acid. Reactions were allowed to proceed for 1 hr at 37° C. on afluorescent plate reader (BioTek) with fluorescent kinetics measuredevery 5 min.

Lentivirus Preparation and Processing

Lentivirus preparation and processing was based on the previously knownmethods. Briefly, 10 μg pSB700 derivatives that include a Zika or DengueRNA fragment, 7.5 μg psPAX2, and 2.5 μg pMD2.G were transfected toHEK293FT cells (Life Technologies, R7007) using the HeBS—CaCl2 method.28 hr after changing media, DMEM supplemented with 10% FBS, 1%penicillin-streptomycin and 4 mM GlutaMAX (ThermoFisher Scientific), thesupernatant was filtered using a 0.45 μm syringe filter. ViralBindLentivirus Purification Kit (Cell Biolabs, VPK-104) and Lenti-XConcentrator (Clontech, 631231) were used to purify and preparelentiviruses from the supernatant. Viral concentration was quantifiedusing QuickTiter Lentivirus Kit (Cell Biolabs, VPK-112). Viral sampleswere spiked into 7% human serum (Sigma, H4522), were heated to 95° C.for 2 min and were used as input to RPA.

Isolation and cDNA Purification of Zika Human Serum Samples

Suspected Zika positive human serum or urine samples were inactivatedwith AVL buffer (Qiagen) and isolation of RNA was achieved with QIAampViral RNA minikit (Qiagen). Isolated RNA was converted into cDNA bymixing random primers, dNTPs, and sample RNA followed by heatdenaturation for 7 minutes at 70° C. Denatured RNA was then reversetranscribed with Superscript III (Invitrogen) incubating at 22-25° C.for 10 minutes, 50° C. for 45 minutes, 55° C. for 15 minutes, and 80° C.for 10 minutes. cDNA was then incubated for 20 minutes at 37° C. withRNAse H (New England Biolabs) to destroy RNA in the RNA:cDNA hybrids.

Genomic DNA Extraction from Human Saliva

2 mL of saliva was collected from volunteers, who were restricted fromconsuming food or drink 30 minutes prior to collection. Samples werethen processed using QIAamp® DNA Blood Mini Kit (Qiagen) as recommendedby the kit protocol. For boiled saliva samples, 400 μL of phosphatebuffered saline (Sigma) was added to 100 μL of volunteer saliva andcentrifuged for 5 min at 1800 g. The supernatant was decanted and thepellet was resuspended in phosphate buffered saline with 0.2% TritonX-100 (Sigma) before incubation at 95° C. for 5 min. 1 μL of sample wasused as direct input into RPA reactions.

Freeze-Drying and Paper Deposition

A glass fiber filter paper (Whatman, 1827-021) was autoclaved for 90 min(Consolidated Stills and Sterilizers, MKII) and was blocked in 5%nuclease-free BSA (EMD Millipore, 126609-10GM) overnight. After rinsingthe papers once with nuclease-free water (Life technologies, AM9932),they were incubated with 4% RNAsecure™ (Life technologies, AM7006) at60° C. for 20 min and were rinsed three more times with thenuclease-free water. Treated papers were dried for 20 min at 80° C. on ahot plate (Cole-Parmer, IKA C-Mag HS7) prior to use. 1.8 μL of C2c2reaction mixture as indicated earlier was put onto the disc (2 mm) thatwas placed in black, clear bottom 384-well plate (Corning, 3544). Forthe freeze-dried test, the plate containing reaction mixture discs wasflash frozen in liquid nitrogen and was freeze-dried overnight asdescribed in Pardee et al (2). RPA samples were diluted 1:10 innuclease-free water, and 1.8 μL of the mixture was loaded onto the paperdiscs and incubated at 37° C. using a plate reader (BioTek Neo).

Bacterial Genomic DNA Extraction

For experiments involving CRE detection, bacterial cultures were grownin lysogeny broth (LB) to mid-log phase, then pelleted and subjected togDNA extraction and purification using the Qiagen DNeasy Blood andTissue Kit, using the manufacturer's protocol for either Gram negativeor Gram positive bacteria, as appropriate. gDNA was quantified by theQuant-It dsDNA assay on a Qubit fluorometer and its quality assessed via200-300 nm absorbance spectrum on a Nanodrop spectrophotometer.

For experiments discriminating between E. coli and P. aeruginosa,bacterial cultures were grown to early stationary phase in Luria-Bertani(LB) broth. 1.0 mL of both E. coli and P. aeruginosa were processedusing the portable PureLyse bacteria gDNA extraction kit (ClaremontBioSolutions). 1× binding buffer was added to the bacterial culturebefore passing through the battery-powered lysis cartridge for threeminutes. 0.5× binding buffer in water was used as a wash solution beforeeluting with 150 μL of water.

Digital Droplet PCR Quantification

To confirm the concentration of ssDNA 1 and ssRNA 1, standard dilutionswere used. Applicant performed digital-droplet PCR (ddPCR). For DNAquantification, droplets were made using the ddPCR Supermix for Probes(no dUTP) with PrimeTime qPCR probes/primer assays designed to targetthe ssDNA 1 sequence. For RNA quantification, droplets were made usingthe one-step RT-ddPCR kit for probes with PrimeTime qPCR probes/primerassays designed to target the ssRNA 1 sequence. Droplets were generatedin either case using the QX200 droplet generator (BioRad) andtransferred to a PCR plate. Droplet-based amplification was performed ona thermocycler as described in the kit's protocol and nucleic acidconcentrations were subsequently determined via measurement on a QX200droplet reader.

Synthetic Standards for Human Genotyping

To create standards for accurate calling of human sample genotypes,Applicant designed primers around the SNP target to amplify ˜200 bpregions from human genomic DNA representing each of the two homozygousgenotypes. The heterozygous standard was then made by mixing thehomozygous standards in a 1:1 ratio. These standards were then dilutedto equivalent genome concentrations (˜0.56 fg/μL) and used as input forSHERLOCK alongside real human samples.

Detection of Tumor Mutant Cell Free-DNA (cfDNA)

Mock cfDNA standards simulating actual patient cfDNA samples werepurchased from a commercial vendor (Horizon Discovery Group). Thesestandards were provided as four allelic fractions (100% WT and 0.1%, 1%,and 5% mutant) for both the BRAF V600E and EGFR L858R mutants. 3 μL ofthese standards were provided as input to SHERLOCK

Analysis of Fluorescence Data

To calculate background subtracted fluorescence data, the initialfluorescence of samples was subtracted to allow for comparisons betweendifferent conditions. Fluorescence for background conditions (either noinput or no crRNA conditions) were subtracted from samples to generatebackground subtracted fluorescence.

Guide ratios for SNP or strain discrimination were calculated bydividing each guide by the sum of guide values, to adjust forsample-to-sample overall variation. crRNA ratios for SNP or straindiscrimination were calculated to adjust for sample-to-sample overallvariation as follows:

${{crRNA}\mspace{14mu} A_{i\mspace{14mu}}{ratio}} = \frac{\left( {m + n} \right)A_{i}}{{\sum\limits_{i = 1}^{m}A_{i}} + {\sum\limits_{i = 1}^{n}B_{i}}}$

where Ai and Bi refer to the SHERLOCK intensity values for technicalreplicate i of the crRNAs sensing allele A or allele B, respectively,for a given individual. Since an assay typically has four technicalreplicates per crRNA, m and n are equal to 4 and the denominator isequivalent to the sum of all eight of the crRNA SHERLOCK intensityvalues for a given SNP locus and individual. Because there are twocrRNAs, the crRNA ratio average across each of the crRNAs for anindividual will always sum to two. Therefore, in the ideal case ofhomozygosity, the mean crRNA ratio for the positive allele crRNA will betwo and the mean crRNA ratio for the negative allele crRNA will be zero.In the ideal case of heterozygosity, the mean crRNA ratio for each ofthe two crRNAs will be one.

Characterization of LwCas13a Cleavage Requirements.

The protospacer flanking site (PFS) is a specific motif present near thetarget site that is required for robust ribonuclease activity by Cas13a.The PFS is located at the 3′ end of the target site and was previouslycharacterized for LshCas13a by our group as H (not G) (1). Although thismotif is akin to a protospacer adjacent motif (PAM), a sequencerestriction for DNA targeting Class 2 systems, it is functionallydifferent as it not involved in preventing self targeting of CRISPR lociin endogenous systems. Future structural studies of Cas13a will likelyelucidate the importance of the PFS for Cas13a:crRNA target complexformation and cleavage activity.

Applicant purified the recombinant LwCas13a protein from E. coli (FIGS.2D-2E) and assayed its ability to cleave a 173-nt ssRNA with eachpossible protospacer flanking site (PFS) nucleotide (A, U, C or G) (FIG.2F). Similar to LshCas13a, LwCas13a can robustly cleave a target with A,U, or C PFS, with less activity on the ssRNA with a G PFS. Althoughweaker activity against ssRNA 1 with a G PFS was observed, Applicantstill saw robust detection for the two target sites with G PFS motifs(Table 3; rs601338 crRNA and Zika targeting crRNA 2). It is likely thatthe H PFS is not required under every circumstance and that in manycases strong cleavage or collateral activity can be achieved with a GPFS.

Discussion of Recombinase Polymerase Amplification (RPA) and OtherIsothermal Amplification Strategies.

Recombinase polymerase amplification (RPA) is an isothermalamplification technique consisting of three essential enzymes: arecombinase, single-stranded DNA-binding proteins (SSBs), and a stranddisplacing polymerase. RPA overcomes many technical difficulties presentin other amplification strategies, particularly polymerase chainreaction (PCR), by not requiring temperature regulation as the enzymesall operate at a constant temperature around 37° C. RPA replacestemperature cycling for global melting of the double-stranded templateand primer annealing with an enzymatic approach inspired by in vivo DNAreplication and repair. Recombinase-primer complexes scandouble-stranded DNA and facilitate strand exchange at complementarysites. The strand exchange is stabilized by SSBs, allowing the primer tostay bound. Spontaneous disassembly of the recombinase occurs in itsADP-bound state, allowing a strand-displacing polymerase to invade andextend the primer, allowing amplification without complexinstrumentation unavailable in point-of-care and field settings. Cyclicrepetition of this process in a temperate range of 37-42° C. results inexponential DNA amplification. The original formulation published usesthe Bacillus subtilis Pol I (Bsu) as the strand-displacing polymerase,T4 uvsX as the recombinase, and T4 gp32 as the single-stranded DNAbinding protein (2), although it is unclear what components are in thecurrent formulation sold by TwistDx used in this study.

Additionally, RPA has a number of limitations:

1) Although Cas13a detection is quantitative (FIG. 15), real-time RPAquantitation can be difficult because of its rapid saturation when therecombinase uses all available ATP. While real-time PCR is quantitativebecause of the ability to cycle amplification, RPA has no mechanism totightly control the rate of amplification. Certain adjustments can bemade to reduce amplification speed, such as reducing available magnesiumor primer concentrations, lowering the reaction temperature, ordesigning inefficient primers. Although some instances of quantitativeSHERLOCK are observed, such as in FIGS. 31A-31D, 32A, 32B, 52A, and 52B,it is not always the case and may depend on the template.2) RPA efficiency can be sensitive to primer design. The manufacturertypically recommends designing longer primers to ensure efficientrecombinase binding with average GC content (40-60%) and screening up to100 primer pairs to find highly sensitive primer pairs. Applicant hasfound with SHERLOCK that only two primer pairs have to be designed toachieve an attomolar test with single molecule sensitivity. Thisrobustness is likely due to the additional amplification of signal byconstitutively active Cas13a collateral activity that offsets anyinefficiencies in amplicon amplification. This quality is particularlyimportant for our bacterial pathogen identification in FIGS. 34A-34C.Issues were experienced with amplifying highly structured regions suchas the 16S rRNA gene sites in bacterial genomes because there is nomelting step involved in RPA. Thus, secondary structure in primersbecomes an issue, limiting amplification efficiency and thussensitivity. The embodiments disclosed herein were believed to besuccessful despite these RPA-specific issues because of additionalsignal amplification from Cas13 a.3) The amplification sequence length must be short (100-200 bp) forefficient RPA. For most applications, this is not a significant issueand perhaps is even advantageous (e.g. cfDNA detection where averagefragment size is 160 bp). Sometimes large amplicon lengths areimportant, such as when universal primers are desired for bacterialdetection and the SNPs for discrimination are spread over a large area.

SHERLOCK's modularity allows any amplification technique, evennon-isothermal approaches, to be used prior to T7 transcription andCas13a detection. This modularity is enabled by the compatibility of theT7 and Cas13a steps in a single reaction allowing detection to beperformed on any amplified DNA input that has a T7 promoter. Prior tousing RPA, nucleic acid sequence based amplification (NASBA) (3, 4) wasattempted for our detection assay (FIG. 10). However NASBA did notdrastically improve the sensitivity of Cas13a (FIGS. 11 and 53). Otheramplification techniques that could be employed prior to detectioninclude PCR, loop mediated isothermal amplification (LAMP) (5), stranddisplacement amplification (SDA) (6), helicase-dependent amplification(HDA) (7), and nicking enzyme amplification reaction (NEAR) (8). Theability to swap any isothermal technique allows SHERLOCK to overcome thespecific limitations of any one amplification technique.

Design of Engineered Mismatches.

Applicant demonstrates that LshCas13a target cleavage was reduced whenthere were two or more mismatches in the target:crRNA duplex but wasrelatively unaffected by single mismatches, an observation Applicantconfirmed for LwCas13a collateral cleavage (FIG. 36A). Applicanthypothesized that by introducing an additional mutation in the crRNAspacer sequence, Applicant would destabilize collateral cleavage againsta target with an additional mismatch (two mismatches in total) whileretaining on-target collateral cleavage, as there would only be a singlemismatch. To test the possibility of engineering increased specificity,Applicant designed multiple crRNAs targeting ssRNA 1 and includedmismatches across the length of the crRNA (FIG. 36A) to optimizeon-target collateral cleavage and minimize collateral cleavage of atarget that differs by a single mismatch. Applicant observed that thesemismatches did not reduce collateral cleavage of ssRNA 1, butsignificantly decreased signal for a target that included an additionalmismatch (ssRNA 2). The designed crRNA that best distinguished betweenssRNA 1 and 2 included synthetic mismatches close to the ssRNA 2mismatch, in effect creating a “bubble,” or distortion in the hybridizedRNA. The loss of sensitivity caused by the coordination of a syntheticmismatch and an additional mismatch present in the target (i.e., adouble mismatch) agrees with the sensitivity of LshCas13a and LwCas13ato consecutive or nearby double mismatches and presents a basis forrational design of crRNAs that enable single-nucleotide distinction(FIG. 36B).

For mismatch detection of ZIKV and DENV strains, our full-length crRNAcontained two mismatches (FIG. 37A, 37B). Due to high sequencedivergence between strains, Applicant was unable to find a continuousstretch of 28 nt with only a single nucleotide difference between thetwo genomes. However, Applicant predicted that shorter crRNAs wouldstill be functional, and designed shorter 23nt crRNAs against targets inthe two ZIKV strains that included a synthetic mismatch in the spacersequence and only one mismatch in the target sequence. These crRNAscould still distinguish African and American strains of ZIKV (FIG. 36C).Subsequent testing of 23 nt and 20 nt crRNA show that reductions ofspacer length reduce activity but maintain or enhance the ability todiscriminate single mismatches (FIGS. 57A-57G). To better understand howsynthetic mismatches may be introduced to facilitate single-nucleotidemutation discrimination, Applicant tiled the synthetic mismatch acrossthe first seven positions of the spacer at three different spacerlengths: 28, 23, and 20 nt (FIG. 57A). On a target with a mutation atthe third position, LwCas13a shows maximal specificity when thesynthetic mismatch is in position 5 of the spacer, with improvedspecificity at shorter spacer lengths, albeit with lower levels ofon-target activity (FIGS. 57B-57G). Applicant also shifted the targetmutation across positions 3-6 and tiled synthetic mismatches in thespacer around the mutation (FIGS. 58A-58C).

Genotyping with SHERLOCK Using Synthetic Standards.

Evaluation of synthetic standards created from PCR amplification of theSNP loci allows for accurate identification of genotypes (FIGS. 60A,60B). By computing all comparisons (ANOVA) between the SHERLOCK resultsof an individual's sample and the synthetic standards, each individual'sgenotype can be identified by finding the synthetic standard that hasthe most similar SHERLOCK detection intensity (FIGS. 60C, 60D). ThisSHERLOCK genotyping approach is generalizable to any SNP locus (FIG.60G).

SHERLOCK is an Affordable, Adaptable CRISPR-Dx Platform.

For the cost analysis of SHERLOCK, reagents determined to be ofnegligible cost were omitted, including DNA templates for the synthesisof crRNA, primers used in RPA, common buffers (MgCl2, Tris HCl,glycerol, NaCl, DTT), glass microfiber filter paper, and RNAsecurereagent. For DNA templates, ultramer synthesis from IDT providesmaterial for 40 in vitro transcription reactions (each being enough for10,000 reactions) for ˜$70, adding negligible cost to crRNA synthesis.For RPA primers, a 25 nmole IDT synthesis of a 30 nt DNA primer can bepurchased for ˜$10, providing material adequate for 5000 SHERLOCKreactions. Glass microfiber paper is available for $0.50/sheet, which issufficient for several hundred SHERLOCK reactions. 4% RNAsecure reagentcosts $7.20/mL, which is sufficient for 500 tests.

In addition, for all experiments, except the paper-based assays,384-well plates were used (Corning 3544), at the cost of$0.036/reaction. Because of the negligible cost, this was not includedin the overall cost analysis. Additionally, SHERLOCK-POC does notrequire the use of a plastic vessel, as it can easily be performed onpaper. The readout method for SHERLOCK used herein was a plate readerequipped with either a filter set or a monochromator. As a capitalinvestment, the cost of the reader was not included in the calculation,as the cost precipitously decreases as more reactions are run on theinstrument and is negligible. For POC applications, cheaper and portablealternatives could be used, such as hand-held spectrophotometers (9) orportable electronic readers (4), which reduce the cost ofinstrumentation to <$200. While these more portable solutions willreduce the speed and ease of readout as compared to bulkier instruments,they allow for more broad use.

Results

The assay and systems described herein may generally comprise a two-stepprocess of amplification and detection. During the first step, thenucleic acid sample, either RNA or DNA, is amplified, for example byisothermal amplification. During the second step, the amplified DNA istranscribed into RNA and subsequently incubated with a CRISPR effector,such as C2c2, and a crRNA programmed to detect the presence of thetarget nucleic acid sequence. To enable detection, a reporter RNA thathas been labeled with a quenched fluorophore is added to the reaction.Collateral cleavage of the reporter RNA results in un-quenching of thefluorophore and allows for real-time detection of the nucleic acidtarget (FIG. 17).

To achieve robust signal detection, an ortholog of C2c2 was identifiedfrom the organism Leptotrichia wadei (LwC2c2) and evaluated. Theactivity of the LwC2c2 protein was evaluated by expressing it along witha synthetic CRISPR array in E. coli and programming it to cleave atarget site within the beta-lactamase mRNA, which leads to death of thebacteria under ampicillin selection (FIG. 2B). Fewer surviving E. colicolonies were observed with the LwC2c2 locus than with the LshC2c2locus, demonstrating a higher cleavage activity of the LwC2c2 ortholog(FIG. 2C). The human-codon optimized LwC2c2 protein was then purifiedfrom E. coli (FIGS. 2D-2E) and its ability to cleave a 173-nt ssRNAassayed with different protospacer flanking site (PFS) nucleotides (FIG.2F). LwC2c2 was able to cleave each of the possible four PFS targets,with slightly less activity on the ssRNA with a G PFS.

Real-time measurement of LwC2c2 RNase collateral activity was measuredusing a commercially available RNA fluorescent plate reader (FIG. 17).To determine the baseline sensitivity of LwC2c2 activity, LwC2c2 wasincubated with ssRNA target 1 (ssRNA 1) and a crRNA that iscomplementary to a site within the ssRNA target, along with the RNAsensor probe (FIG. 18). This yielded a sensitivity of ˜50 fM (FIG. 27A),which, although more sensitive than other recent nucleic acid detectiontechnologies (Pardee et al., 2014), is not sensitive enough for manydiagnostic applications which require sub-femtomolar detectionperformance (Barletta et al., 2004; Emmadi et al., 2011; Rissin et al.,2010; Song et al., 2013).

To increase sensitivity, an isothermal amplification step was addedprior to incubation with LwC2c2. Coupling LwC2c2-mediated detection withpreviously used isothermal amplification approaches such as nucleic acidsequence based amplification (NASBA)(Compton, 1991; Pardee et al., 2016)improved sensitivity to a certain extent (FIG. 11). An alternativeisothermal amplification approach, recombinase polymerase amplification(RPA) (Piepenburg et al., 2006), was tested which can be used to amplifyDNA exponentially in under two hours. By adding a T7 RNA polymerasepromoter onto the RPA primers, amplified DNA can be converted to RNA forsubsequent detection by LwC2c2 (FIG. 17). Thus, in certain exampleembodiments, the assay comprises the combination of amplification byRPA, T7 RNA polymerase conversion of DNA to RNA, and subsequentdetection of the RNA by C2c2 unlocking of fluorescence from a quenchedreporter.

Using the example method on a synthesized DNA version of ssRNA 1, it waspossible to achieve attomolar sensitivity in the range of 1-10 moleculesper reaction (FIG. 27B, left). In order to verify the accuracy ofdetection, the concentration of input DNA was qualified withdigital-droplet PCR and confirmed that the lowest detectable targetconcentration (2 aM) was at a concentration of a single molecule permicroliter. With the addition of a reverse transcription step, RPA canalso amplify RNA into a dsDNA form, allowing us attomolar sensitivity onssRNA 1 to be achieved (27B, right). Similarly, the concentrations ofRNA targets were confirmed by digital-droplet PCR. To evaluate theviability of the example method to function as a POC diagnostic test,the ability of all components—RPA, T7 polymerase amplification, andLwC2c2 detection—to function in a single reaction were tested and foundattomolar sensitivity with a one-pot version of the assay (FIG. 22).

The Assay is Capable of Sensitive Viral Detection in Liquid or on Paper

It was next determined whether the assay would be effective ininfectious disease applications that require high sensitivity and couldbenefit from a portable diagnostic. To test detection in a model system,lentiviruses harboring RNA fragments of the Zika virus genome and therelated flavivirus Dengue (Dejnirattisai et al., 2016) were produced andthe number of viral particles quantified (FIG. 31A). Levels of mockvirus were detected down to 2 aM. At the same time, it was also possibleto show clear discrimination between these proxy viruses containing Zikaand Dengue RNA fragments (FIG. 31B). To determine whether the assaywould be compatible with freeze-drying to remove dependence on coldchains for distribution, the reaction components were freeze-dried.After using the sample to rehydrate the lyophilized components, 20 fM ofssRNA 1 was detected (FIG. 33A). Because resource-poor and POC settingswould benefit from a paper test for ease of usability, the activity ofC2c2 detection on glass fiber paper was also evaluated and found that apaper-spotted C2c2 reaction was capable of target detection (FIG. 33B).In combination, freeze-drying and paper-spotting the C2c2 detectionreaction resulted in sensitive detection of ssRNA 1 (FIG. 33C). Similarlevels of sensitivity were also observed for detection of a syntheticZika viral RNA fragment between LwC2c2 in solution and freeze-driedLwC2c2, demonstrating the robustness of freeze-dried SHERLOCK and thepotential for a rapid, POC Zika virus diagnostic (FIGS. 33D, 33E).Toward this end, the ability of the POC variant of the assay was testedto determine the ability to discriminate Zika RNA from Dengue RNA (FIG.31C). While paper-spotting and lyophilization slightly reduced theabsolute signal of the readout, the assay still significantly detectedmock Zika virus at concentrations as low as 20 aM (FIG. 31D), comparedto detection of mock virus with the Dengue control sequence.

Zika viral RNA levels in humans have been reported to be as low as 3×10⁶copies/mL (4.9 fM) in patient saliva and 7.2×10⁵ copies/mL (1.2 fM) inpatient serum (Barzon et al., 2016; Gourinat et al., 2015; Lanciotti etal., 2008). From obtained patient samples, concentrations as low as1.25×10³ copies/mL (2.1 aM) were observed. To evaluate whether the assayis capable of Zika virus detection of low-titer clinical isolates, viralRNA was extracted from patients and reverse transcribed and theresulting cDNA was used as input for the assay (FIG. 32A). Significantdetection for the Zika human serum samples was observed atconcentrations down to 1.25 copy/uL (2.1 aM) (FIG. 32B). Furthermore,signal from patient samples was predictive of Zika viral RNA copy numberand could be used to predict viral load (FIGS. 31A-31D). To test broadapplicability for disease situations where nucleic acid purification isunavailable, detection of ssRNA 1 spiked into human serum was tested,and it was determined that the assay was activated at serum levels below2% (FIG. 33G).

Bacterial Pathogen Distinction and Gene Distinction

To determine if the assay could be used to distinguish bacterialpathogens, the 16S V3 region was selected as an initial target, as theconserved flanking regions allow universal RPA primers to be used acrossbacterial species, and the variable internal region allowing fordifferentiation of species. A panel of 5 possible targeting crRNAs weredesigned for pathogenic strains and isolated E. coli and Pseudomonasaeruginosa gDNA (FIG. 34A). The assay was capable of distinguishing E.coli or P. aeruginosa gDNA and showed low background signal for crRNAsof other species (FIGS. 34A-34C).

The assay can also be adapted to rapidly detect and distinguishbacterial genes of interest, such as antibiotic-resistance genes.Carbapenem-resistant enterobacteria (CRE) are a significant emergingpublic health challenge (Gupta et al., 2011). The ability of the assayto detect carbapenem-resistance genes was evaluated, and if the testcould distinguish between different carbapenem-resistance genes.Klebsiella pneumonia was obtained from clinical isolates harboringeither Klebsiella pneumoniae carbapenemase (KPC) or New Delhimetallo-beta-lactamase 1 (NDM-1) resistance genes and designed crRNAs todistinguish between the genes. All CRE had significant signal overbacteria lacking these resistance genes (FIG. 35A) and that we couldsignificantly distinguish between KPC and NDM-1 strains of resistance(FIG. 35B).

Single-Base Mismatch Specificity of CRISPR RNA-Guided RNases

It has been shown that certain CRISPR RNA-guided RNase orthologues, suchas LshC2c2, do not readily distinguish single-base mismatches.(Abudayyeh et al., 2016). As demonstrated herein, LwC2c2 also sharesthis feature (FIG. 37A). To increase the specificity of LwC2c2 cleavage,a system for introducing synthetic mismatches in the crRNA:target duplexwas developed that increases the total sensitivity to mismatches andenables single-base mismatch sensitivity. Multiple crRNAs for target 1were designed and included mismatches across the length of the crRNA(FIG. 37A) to optimize on-target cleavage and minimize cleavage of atarget that differs by a single mismatch. These mismatches did notreduce cleavage efficiency of ssRNA target 1, but significantlydecreased signal for a target that included an additional mismatch(ssRNA target 2). The designed crRNA that best distinguished betweentargets 1 and 2 included synthetic mismatches close to the target 2mismatch, in effect creating a “bubble.” The loss of sensitivity causedby the coordination of a synthetic mismatch and an additional mismatchpresent in the target (i.e., a double mismatch) agrees with thesensitivity of LshC2c2 to consecutive or nearby double mismatches(Abudayyeh et al., 2016) and presents a format for rational design ofcrRNAs that enable single-nucleotide distinction (FIG. 37B).

Having demonstrated that C2c2 can be engineered to recognize single-basemismatches, it was determined whether this engineered specificity couldbe used to distinguish between closely related viral pathogens. MultiplecrRNAs were designed to detect either the African or American strains ofZika virus (FIG. 37A) and either strain 1 or 3 of Dengue virus (FIG.37C). These crRNAs included a synthetic mismatch in the spacer sequence,causing a single bubble to form when duplexed to the on-target straindue to the synthetic mismatch. However, when the synthetic mismatchspacer is duplexed to the off-target strain two bubbles form due to thesynthetic mismatch and the SNP mismatch. The synthetic mismatch crRNAsdetected their corresponding strains with significantly higher signalthan the off-target strain allowing for robust strain distinction (FIGS.37B, 37D). Due to the significant sequence similarity between strains,it was not possible to find a continuous stretch of 28 nt with only asingle nucleotide difference between the two genomes in order todemonstrate true single-nucleotide strain distinction. However, it waspredicted that shorter crRNAs would still be functional, as they arewith LshC2c2 (Abudayyeh et al., 2016), and accordingly shorter 23-ntcrRNAs were designed against targets in the two Zika strains thatincluded a synthetic mismatch in the spacer sequence and only onemismatch in the target sequence. These crRNAs were still capable ofdistinguishing the African and American strains of Zika with highsensitivity (FIG. 36C).

Rapid Genotyping Using DNA Purified from Saliva

Rapid genotyping from human saliva could be useful in emergencypharmacogenomic situations or for at-home diagnostics. To demonstratethe potential of the embodiments disclosed herein for genotyping, fiveloci were chosen to benchmark C2c2 detection using 23andMe genotypingdata as the gold standard (Eriksson et al., 2010) (FIG. 38A). The fiveloci span a broad range of functional associations, includingsensitivity to drugs, such as statins or acetaminophen, norovirussusceptibility, and risk of heart disease (Table 7).

TABLE 7 SNP Variants tested ID Gene Category rs5082 APOA2 Saturated fatconsumption and weight gain rs1467558 CD44 Acetaminophen metabolismrs2952768 near CREB1 morphine dependence rs4363657 SLCO1B1 4.5x increasemyopathy risk for statin users rs601338 FUT2 resistance to norovirus

Saliva from four human subjects was collected and the genomic DNApurified using a simple commercial kit in less than an hour. The foursubjects had a diverse set of genotypes across the five loci, providinga wide enough sample space for which to benchmark the assay forgenotyping. For each of the five SNP loci, a subject's genomic DNA wasamplified using RPA with the appropriate primers followed by detectionwith LwC2c2 and pairs of crRNAs designed to specifically detect one ofthe two possible alleles (FIG. 38B). The assay was specific enough todistinguish alleles with high significance and to infer both homozygousand heterozygous genotypes. Because a DNA extraction protocol wasperformed on the saliva prior to detection, the assay was tested todetermine if it could be made even more amenable for POC genotyping byusing saliva heated to 95° C. for 5 minutes without any furtherextraction. The assay was capable of correctly genotyping two patientswhose saliva was only subjected to heating for 5 minutes and thensubsequent amplification and C2c2 detection (FIG. 40B).

Detection of Cancerous Mutations in cfDNA at Low-Allelic Fractions

Because the assay is highly specific to single nucleotide differences intargets, a test was devised to determine if the assay was sensitiveenough to detect cancer mutations in cell-free DNA (cfDNA). cfDNAfragments are small percentage (0.1% to 5%) of wild-type cfDNA fragments(Bettegowda et al., 2014; Newman et al., 2014; Olmedillas Lopez et al.,2016; Qin et al., 2016). A significant challenge in the cfDNA field isdetecting these mutations because they are typically difficult todiscover given the high levels of non-mutated DNA found in thebackground in blood (Bettegowda et al., 2014; Newman et al., 2014; Qinet al., 2016). A POC cfDNA cancer test would also be useful for regularscreening of cancer presence, especially for patients at risk forremission.

The assay's ability to detect mutant DNA in wild-type background wasdetermined by diluting dsDNA target 1 in a background of ssDNA1 with asingle mutation in the crRNA target site (FIGS. 41A, 41B). LwC2c2 wascapable of sensing dsDNA 1 to levels as low as 0.1% of the backgrounddsDNA and within attomolar concentrations of dsDNA 1. This result showsthat LwC2c2 cleavage of background mutant dsDNA 1 is low enough to allowrobust detection of the on-target dsDNA at 0.1% allelic fraction. Atlevels lower than 0.1%, background activity is likely an issue,preventing any further significant detection of the correct target.

Because the assay could sense synthetic targets with allelic fractionsin a clinically relevant range, it was evaluated whether the assay wascapable of detecting cancer mutations in cfDNA. RPA primers to twodifferent cancer mutations, EGFR L858R and BRAF V600E, were designed andcommercial cfDNA standards were used with allelic fractions of 5%, 1%,and 0.1% that resemble actual human cfDNA samples to test. Using a pairof crRNAs that could distinguish the mutant allele from the wild-typeallele (FIG. 38C), detection of the 0.1% allelic fraction for both ofthe mutant loci was achieved (FIGS. 39A, 39B).

Discussion

By combining the natural properties of C2c2 with isothermalamplification and a quenched fluorescent probe, the assay and systemsdisclosed herein have been demonstrated as a versatile, robust method todetect RNA and DNA, and suitable for a variety of rapid diagnosesincluding infectious disease applications and rapid genotyping. A majoradvantage of the assays and systems disclosed herein is that a new POCtest can be redesigned and synthesized in a matter of days for as low as$0.6/test.

Because many human disease applications require the ability to detectsingle mismatches a rational approach was developed to engineer crRNAsto be highly specific to a single mismatch in the target sequence byintroducing a synthetic mismatch in the spacer sequence of the crRNA.Other approaches for achieving specificity with CRISPR effectors rely onscreening-based methods over dozens of guide designs (Chavez et al.,2016). Using designed mismatch crRNAs, discrimination of Zika and Dengueviral strains in sites that differ by a single mismatch, rapidgenotyping of SNPs from human saliva gDNA, and detection of cancermutations in cfDNA samples, was demonstrated.

The low cost and adaptability of the assay platform lends itself tofurther applications including (i) general RNA/DNA quantitationexperience in substitute of specific qPCR assays, such as Taqman, (ii)rapid, multiplexed RNA expression detection resembling microarrays, and(iii) other sensitive detection applications, such as detection ofnucleic acid contamination from other sources in food. Additionally,C2c2 could potentially be used for detection of transcripts withinbiological settings, such as in cells, and given the highly specificnature of C2c2 detection, it may be possible to track allelic specificexpression of transcripts or disease-associated mutations in live cells.With the wide availability of aptamers, it might also be possible tosense proteins by coupling the detection of protein by an aptamer to therevealing of a cryptic amplification site for RPA followed by C2c2detection.

Nucleic Acid Detection with CRISPR-Cas13a/C2c2: Attomolar Sensitivityand Single Nucleotide Specificity

To achieve robust signal detection, Applicant identified an ortholog ofCas13a from Leptotrichia wadei (LwCas13a), which displays greaterRNA-guided RNase activity relative to Leptotrichia shahii Cas13a(LshCas13a) (10) (FIGS. 2A-2F, see also above “Characterization ofLwCas13a cleavage requirements”). LwCas13a incubated with ssRNA target 1(ssRNA 1), crRNA, and reporter (quenched fluorescent RNA) (FIG. 18) (13)yielded a detection sensitivity of ˜50 fM (FIG. 51, 15), which is notsensitive enough for many diagnostic applications (12, 14-16). Applicanttherefore explored combining Cas13a-based detection with differentisothermal amplification steps (FIG. 10, 11, 53, 16) (17, 18). Of themethods explored, recombinase polymerase amplification (RPA) (18)afforded the greatest sensitivity and can be coupled with T7transcription to convert amplified DNA to RNA for subsequent detectionby LwCas13a (see also above “Discussion of Recombinase PolymeraseAmplification (RPA) and other isothermal amplification strategies.”).Applicant refer to this combination of amplification by RPA, T7 RNApolymerase transcription of amplified DNA to RNA, and detection oftarget RNA by Cas13a collateral RNA cleavage-mediated release ofreporter signal as SHERLOCK.

Applicant first determined the sensitivity of SHERLOCK for detection ofRNA (when coupled with reverse transcription) or DNA targets. Applicantachieved single molecule sensitivity for both RNA and DNA, as verifiedby digital-droplet PCR (ddPCR) (FIGS. 27A, 27B, 51, 54A, 54B). Attomolarsensitivity was maintained when all SHERLOCK components were combined ina single reaction, demonstrating the viability of this platform as apoint-of-care (POC) diagnostic (FIG. 54C). SHERLOCK has similar levelsof sensitivity as ddPCR and quantitative PCR (qPCR), two establishedsensitive nucleic acid detection approaches, whereas RPA alone was notsensitive enough to detect low levels of target (FIGS. 55A-55D).Moreover, SHERLOCK shows less variation than ddPCR, qPCR, and RPA, asmeasured by the coefficient of variation across replicates (FIGS. 55E,55F).

Applicant next examined whether SHERLOCK would be effective ininfectious disease applications that require high sensitivity. Applicantproduced lentiviruses harboring genome fragments of either Zika virus(ZIKV) or the related flavivirus Dengue (DENV) (19) (FIG. 31A). SHERLOCKdetected viral particles down to 2 aM and could discriminate betweenZIKV and DENV (FIG. 31B). To explore the potential use of SHERLOCK inthe field, Applicant first demonstrated that Cas13acrRNA complexeslyophilized and subsequently rehydrated (20) could detect 20 fM ofnonamplified ssRNA 1 (FIG. 33A) and that target detection was alsopossible on glass fiber paper (FIG. 33B). The other components ofSHERLOCK are also amenable to freeze-drying: RPA is provided as alyophilized reagent at ambient temperature, and Applicant previouslydemonstrated that T7 polymerase tolerates freeze-drying (2). Incombination, freeze-drying and paper-spotting the Cas13a detectionreaction resulted in comparable levels of sensitive detection of ssRNA 1as aqueous reactions (FIGS. 33C-33E). Although paper-spotting andlyophilization slightly reduced the absolute signal of the readout,SHERLOCK (FIG. 31C) could readily detect mock ZIKV virus atconcentrations as low as 20 aM (FIG. 31D). SHERLOCK is also able todetect ZIKV in clinical isolates (serum, urine, or saliva) where titerscan be as low as 2×103 copies/mL (3.2 aM) (21). ZIKV RNA extracted frompatient serum or urine samples and reverse transcribed into cDNA (FIGS.32A and 52A) could be detected at concentrations down to 1.25×103copies/mL (2.1 aM), as verified by qPCR (FIGS. 32B and 52B).Furthermore, the signal from patient samples was predictive of ZIKV RNAcopy number and could be used to predict viral load (FIG. 33F). Tosimulate sample detection without nucleic acid purification, Applicantmeasured detection of ssRNA 1 spiked into human serum, and found thatCas13a could detect RNA in reactions containing as much as 2% serum(FIG. 33G). Another important epidemiological application for theembodiments disclosed herein is the identification of bacterialpathogens and detection of specific bacterial genes. Applicant targetedthe 16S rRNA gene V3 region, where conserved flanking regions allowuniversal RPA primers to be used across bacterial species and thevariable internal region allows for differentiation of species. In apanel of five possible targeting crRNAs for different pathogenic strainsand gDNA isolated from E. coli and Pseudomonas aeruginosa (FIG. 34A),SHERLOCK correctly genotyped strains and showed low cross-reactivity(FIGS. 34B, 34C). Additionally, Applicant was able to use SHERLOCK todistinguish between clinical isolates of Klebsiella pneumoniae with twodifferent resistance genes: Klebsiella pneumoniae carbapenemase (KPC)and New Delhi metallo-beta-lactamase 1 (NDM-1) (22) (FIG. 56).

To increase the specificity of SHERLOCK, Applicant introduced syntheticmismatches in the crRNA:target duplex that enable LwCas13a todiscriminate between targets that differ by a single-base mismatch(FIGS. 36A, 36B; see also above “Design of Engineered Mismatches”).Applicant designed multiple crRNAs with synthetic mismatches in thespacer sequences to detect either the African or American strains ofZIKV (FIG. 37A) and strain 1 or 3 of DENV (FIG. 37C). Synthetic mismatchcrRNAs detected their corresponding strains with significantly highersignal (two-tailed Student t-test; p<0.01) than the off-target strain,allowing for robust strain discrimination based off single mismatches(FIGS. 37B, 37D, 36C). Further characterization revealed that Cas13adetection achieves maximal specificity while maintaining on-targetsensitivity when a mutation is in position 3 of the spacer and thesynthetic mismatch is in position 5 (FIGS. 57A-57G and 58A-58C). Theability to detect single-base differences opens the opportunity of usingSHERLOCK for rapid human genotyping. Applicant chose five loci spanninga range of health-related single-nucleotide polymorphisms (SNPs)(Table 1) and benchmarked SHERLOCK detection using 23andMe genotypingdata as the gold standard at these SNPs (23) (FIG. 38A). Applicantcollected saliva from four human subjects with diverse genotypes acrossthe loci of interest, and extracted genomic DNA either throughcommercial column purification or direct heating for five minutes (20).SHERLOCK distinguished alleles with high significance and with enoughspecificity to infer both homozygous and heterozygous genotypes (FIG.38B, 40A, 40B, 59, 60A-60E; see also above “Genotyping with SHERLOCKusing synthetic standards”). Finally, Applicant sought to determine ifSHERLOCK could detect low frequency cancer mutations in cell free (cf)DNA fragments, which is challenging because of the high levels ofwild-type DNA in patient blood (24-26). Applicant first found thatSHERLOCK could detect ssDNA 1 at attomolar concentrations diluted in abackground of genomic DNA (FIG. 61). Next, Applicant found that SHERLOCKwas also able to detect single nucleotide polymorphism (SNP)-containingalleles (FIG. 41A, 41B) at levels as low as 0.1% of background DNA,which is in the clinically relevant range. Applicant then demonstratedthat SHERLOCK could detect two different cancer mutations, EGFR L858Rand BRAF V600E, in mock cfDNA samples with allelic fractions as low as0.1% (FIGS. 38A-38D and 39A-39B) (20).

The SHERLOCK platform lends itself to further applications including (i)general RNA/DNA quantitation in lieu of specific qPCR assays, such asTaqMan, (ii) rapid, multiplexed RNA expression detection, and (iii)other sensitive detection applications, such as detection of nucleicacid contamination. Additionally, Cas13a could potentially detecttranscripts within biological settings and track allele-specificexpression of transcripts or disease-associated mutations in live cells.SHERLOCK is a versatile, robust method to detect RNA and DNA, suitablefor rapid diagnoses including infectious disease applications andsensitive genotyping. A SHERLOCK paper test can be redesigned andsynthesized in a matter of days for as low as $0.61/test (see also above“SHERLOCK is an affordable, adaptable CRISPR-Dx platform”) withconfidence, as almost every crRNA tested resulted in high sensitivityand specificity. These qualities highlight the power of CRISPR-Dx andopen new avenues for rapid, robust and sensitive detection of biologicalmolecules.

TABLE 8 RPA Primers used Name Sequence 1st Fig. RP0683-RPA ssDNA/ssRNA 1F (SEQ. I.D. No. 18) FIG. 27B RP0684-RPA ssDNA/ssRNA 1 R (SEQ. I.D. No.19) FIG. 27B AMPL-25 Zika 8B long-rpa3-f (SEQ. I.D. No. 20) FIG. 31BAMPL-26 Zika 8B long-rpa3-r (SEQ. I.D. No. 21) FIG. 31B RP819-zikaregion 8 F (SEQ. I.D. No. 22) FIG. 31C RP821-zika region 8 R (SEQ. I.D.No. 23) FIG. 31C 517 bacterial V3 F (SEQ. I.D. No. 24) FIG. 34B and 34CRP758 bacterial V3 R (SEQ. I.D. No. 25) FIG. 34B and 34C wR0074 A2rs5082 F (SEQ. I.D. No. 26) FIG. 38B wR0074 E2 rs5082 R AA (SEQ. I.D.No. 27) FIG. 38B wR0074 A4 rs1467558 F (SEQ. I.D. No. 28) FIG. 38BwR0074 E4 rs1467558 R (SEQ. I.D. No. 29) FIG. 38B wR0074 A5 rs2952768 F(SEQ. I.D. No. 30) FIG. 38B wR0074 E5 rs2952768 R (SEQ. I.D. No. 31)FIG. 38B wR0074 A9 rs4363657 F (SEQ. I.D. No. 32) FIG. 38B wR0074 E9rs4363657 R (SEQ. I.D. No. 33) FIG. 38B wR0074 A11 rs601338 F (SEQ. I.D.No. 34) FIG. 38B wR0074 E11 rs601338 R (SEQ. I.D. No. 35) FIG. 38B RP824BRAFV600E cfDNA F (SEQ. I.D. No. 36) FIG. 39A RP769 BRAFV600E cfDNA R(SEQ. I.D. No. 37) FIG. 39A RP826 EGFR858R cfDNA F (SEQ. I.D. No. 38)FIG. 39B RP804 EGFR858R cfDNA R (SEQ. I.D. No. 39) FIG. 39B AMPL-31T1-nasba1-f (SEQ. I.D. No. 40) FIG. 11 AMPL-32 T1-nasba1-r (SEQ. I.D.No. 41) FIG. 11 AMPL-33 T1-nasba2-f (SEQ. I.D. No. 42) FIG. 11 AMPL-34T1-nasba2-r (SEQ. I.D. No. 43) FIG. 11 AMPL-35 T1-nasba3-f (SEQ. I.D.No. 44) FIG. 11 AMPL-36 T1-nasba3-r (SEQ. I.D. No. 45) FIG. 11 wR0075 A1KPC F (SEQ. I.D. No. 46) FIG. 35A wR0075 B1 KPC R (SEQ. I.D. No. 47)FIG. 35A wR0075 A3 NDM F (SEQ. I.D. No. 48) FIG. 35A wR0075 B3 NDM R(SEQ. I.D. No. 49) FIG. 35A

TABLE 9 crRNA sequences used Name Complete crRNA sequence Spacersequence 1^(st) Fig. PFS Target 1 crRNA (SEQ. I.D. No. 50) (SEQ. I.D.No. 51) FIG. 2F C Zika targeting (SEQ. I.D. No. 52) (SEQ. I.D. No. 53)FIG. 31A U crRNA 1 Zika targeting (SEQ. I.D. No. 54) (SEQ. I.D. No. 55)FIG. 33D G crRNA 2 E. coli detection (SEQ. I.D. No. 56) (SEQ. I.D. No.57) FIG. 22 U crRNA K. pneumoniae (SEQ. I.D. No. 58) (SEQ. I.D. No. 59)FIG. 34B U detection crRNA and 34C P. aeruginosa (SEQ. I.D. No. 60)(SEQ. I.D. No. 61) FIG. 34B U detection crRNA and 34C M. tuberculosis(SEQ. I.D. No. 62) (SEQ. I.D. No. 63) FIG. 34B U detection crRNA and 34CS. aureus detection (SEQ. I.D. No. 64) (SEQ. I.D. No. 65) FIG. 34B GcrRNA and 34C KPC crRNA (SEQ. I.D. No. 66) (SEQ. I.D. No. 67) FIG. 35A UNDM crRNA (SEQ. I.D. No. 68) (SEQ. I.D. No. 69) FIG. 35A C mismatchcrRNA 1 (SEQ. I.D. No. 70) (SEQ. I.D. No. 71) FIG. 36A C mismatch crRNA2 (SEQ. I.D. No. 72) (SEQ. I.D. No. 73) FIG. 36A C mismatch crRNA 3(SEQ. I.D. No. 74) (SEQ. I.D. No. 75) FIG. 36A C mismatch crRNA 4 (SEQ.I.D. No. 76) (SEQ. I.D. No. 77) FIG. 36A C mismatch crRNA 5 (SEQ. I.D.No. 78) (SEQ. I.D. No. 79) FIG. 36A C mismatch crRNA 6 (SEQ. I.D. No.80) (SEQ. I.D. No. 81) FIG. 36A C mismatch crRNA 7 (SEQ. I.D. No. 82)(SEQ. I.D. No. 83) FIG. 36A C mismatch crRNA 8 (SEQ. I.D. No. 84) (SEQ.I.D. No. 85) FIG. 36A C mismatch crRNA 9 (SEQ. I.D. No. 86) (SEQ. I.D.No. 87) FIG. 36A C mismatch crRNA (SEQ. I.D. No. 88) (SEQ. I.D. No. 89)FIG. 36A C 10 African crRNA 1 (SEQ. I.D. No. 90) (SEQ. I.D. No. 91) FIG.38A C African crRNA 2 (SEQ. I.D. No. 92) (SEQ. I.D. No. 93) FIG. 38A CAmerican crRNA 1 (SEQ. I.D. No. 94) (SEQ. I.D. No. 95) FIG. 38A UAmerican crRNA 2 (SEQ. I.D. No. 96) (SEQ. I.D. No. 97) FIG. 38A U Denguestrain 3 (SEQ. I.D. No. 98) (SEQ. I.D. No. 99) FIG. 38C A crRNA 1 Denguestrain 3 (SEQ. I.D. No. 100) (SEQ. I.D. No. 101) FIG. 38C A crRNA 2Dengue strain 1 (SEQ. I.D. No. 102) (SEQ. I.D. No. 103) FIG. 38C A crRNA1 Dengue strain 1 (SEQ. I.D. No. 104) (SEQ. I.D. No. 105) FIG. 38C AcrRNA 2 Shorter African (SEQ. I.D. No. 106) (SEQ. I.D. No. 107) FIG. 36CC crRNA 1 Shorter African (SEQ. I.D. No. 108) (SEQ. I.D. No. 109) FIG.36C C crRNA 2 Shorter American (SEQ. I.D. No. 110) (SEQ. I.D. No. 111)FIG. 36C U crRNA 1 Shorter American (SEQ. I.D. No. 112) (SEQ. I.D. No.113) FIG. 36C U crRNA 2 rs1467558 crRNA C (SEQ. I.D. No. 114) (SEQ. I.D.No. 115) FIG. 38B C rs1467558 crRNA T (SEQ. I.D. No. 116) (SEQ. I.D. No.117) FIG. 38B C rs2952768 crRNA C (SEQ. I.D. No. 118) (SEQ. I.D. No.119) FIG. 38B A rs2952768 crRNA T (SEQ. I.D. No. 120) (SEQ. I.D. No.121) FIG. 38B A rs4363657 crRNA C (SEQ. I.D. No. 122) (SEQ. I.D. No.123) FIG. 38B A rs4363657 crRNA T (SEQ. I.D. No. 124) (SEQ. I.D. No.125) FIG. 38B A rs601338 crRNA A (SEQ. I.D. No. 126) (SEQ. I.D. No. 127)FIG. 38B G rs601338 crRNA G (SEQ. I.D. No. 128) (SEQ. I.D. No. 129) FIG.38B G rs5082 crRNA G (SEQ. I.D. No. 130) (SEQ. I.D. No. 131) FIG. 40A Ars5082 crRNA A (SEQ. I.D. No. 132) A EGFR L858R (SEQ. I.D. No. 134)(SEQ. I.D. No. 135) FIG. 38C C wild-type crRNA EGFR L858R (SEQ. I.D. No.136) (SEQ. I.D. No. 137) FIG. 38C C mutant crRNA BRAF V600E (SEQ. I.D.No. 138) (SEQ. I.D. No. 139) FIG. 38C A wild-type crRNA BRAF V600E (SEQ.I.D. No. 140) (SEQ. I.D. No. 141) FIG. 38C A mutant crRNA 23 nt mismatch(SEQ. I.D. No. 303) (SEQ. I.D. No. 304) FIG. 57D C crRNA 1 23 ntmismatch (SEQ. I.D. No. 305) (SEQ. I.D. No. 306) FIG. 57D C crRNA 2 23nt mismatch (SEQ. I.D. No. 307) (SEQ. I.D. No. 308) FIG. 57D C crRNA 423 nt mismatch (SEQ. I.D. No. 234) (SEQ. I.D. No. 235) FIG. 57D C crRNA5 23 nt mismatch (SEQ. I.D. No. 236) (SEQ. I.D. No. 237) FIG. 57D CcrRNA 6 23 nt mismatch (SEQ. I.D. No. 238) (SEQ. I.D. No. 239) FIG. 57DC crRNA 7 20 nt mismatch (SEQ. I.D. No. 240) (SEQ. I.D. No. 241) FIG.57F C crRNA 1 20 nt mismatch (SEQ. I.D. No. 242) (SEQ. I.D. No. 243)FIG. 57F C crRNA 2 20 nt mismatch (SEQ. I.D. No. 244) (SEQ. I.D. No.245) FIG. 57F C crRNA 4 20 nt mismatch (SEQ. I.D. No. 246) (SEQ. I.D.No. 247) FIG. 57F C crRNA 5 20 nt mismatch (SEQ. I.D. No. 248) (SEQ.I.D. No. 249) FIG. 57F C crRNA 6 20 nt mismatch (SEQ. I.D. No. 250)(SEQ. I.D. No. 251) FIG. 57F C crRNA 7 target mismatch (SEQ. I.D. No.252) (SEQ. I.D. No. 253) FIG. 58B C 4 mismatch crRNA 1 target mismatch(SEQ. I.D. No. 254) (SEQ. I.D. No. 255) FIG. 58B C 4 mismatch crRNA 2target mismatch (SEQ. I.D. No. 256) (SEQ. I.D. No. 257) FIG. 58B C 4mismatch crRNA 3 target mismatch (SEQ. I.D. No. 258) (SEQ. I.D. No. 259)FIG. 58B C 4 mismatch crRNA 5 target mismatch SEQ. I.D. No. 260) (SEQ.I.D. No. 261) FIG. 58B C 4 mismatch crRNA 6 target mismatch (SEQ. I.D.No. 262) (SEQ. I.D. No. 263) FIG. 58B C 4 mismatch crRNA 7 targetmismatch (SEQ. I.D. No. 264) (SEQ. I.D. No. 265) FIG. 58B C 5 mismatchcrRNA 2 target mismatch (SEQ. I.D. No. 266) (SEQ. I.D. No. 267) FIG. 58BC 5 mismatch crRNA 3 target mismatch (SEQ. I.D. No. 268) (SEQ. I.D. No.269) FIG. 58B C 5 mismatch crRNA 4 target mismatch (SEQ. I.D. No. 270)(SEQ. I.D. No. 271) FIG. 58B C 5 mismatch crRNA 6 target mismatch (SEQ.I.D. No. 272) (SEQ. I.D. No. 273) FIG. 58B C 5 mismatch crRNA 7 targetmismatch (SEQ. I.D. No. 274) (SEQ. I.D. No. 275) FIG. 58B C 5 mismatchcrRNA 8 target mismatch (SEQ. I.D. No. 276) (SEQ. I.D. No. 277) FIG. 58BC 6 mismatch crRNA 3 target mismatch (SEQ. I.D. No. 278) (SEQ. I.D. No.279) FIG. 58B C 6 mismatch crRNA 4 target mismatch (SEQ. I.D. No. 280)(SEQ. I.D. No. 281) FIG. 58B C 6 mismatch crRNA 5 target mismatch (SEQ.I.D. No. 282) (SEQ. I.D. No. 283) FIG. 58B C 6 mismatch crRNA 7 targetmismatch (SEQ. I.D. No. 284) (SEQ. I.D. No. 285) FIG. 58B C 6 mismatchcrRNA 8 target mismatch (SEQ. I.D. No. 286) (SEQ. I.D. No. 287) FIG. 58BC 6 mismatch crRNA 9

TABLE 10 RNA and DNA targets used in this Example Name Sequence 1^(st)Fig ssRNA 1 (C PFS) (SEQ. I.D. No. 288 FIG. 2F ssRNA 1 (G PFS) (SEQ.I.D. No. 289) FIG. 2F ssRNA 1 (A PFS) (SEQ. I.D. No. 290) FIG. 2F ssRNA1 (U PFS) (SEQ. I.D. No. 291) FIG. 2F ssDNA 1 (SEQ. I.D. No. 292) FIG.27A, 27B DNA 2 (SEQ. I.D. No. 293) FIG. 54B ZIKV in lentivirus (SEQ.I.D. No. 294) FIG. 31B DENV in lentivirus (SEQ. I.D. No. 295) FIG. 31BSynthetic ZIKV (SEQ. I.D. No. 296) FIG. 33D target Synthetic African(SEQ. I.D. No. 297) FIG. 37A ZIKV target Synthetic American (SEQ. I.D.No. 298) FIG. 37A ZIKV target Synthetic Dengue (SEQ. I.D. No. 299) FIG.37C strain 1 target Synthetic Dengue (SEQ. I.D. No. 300) FIG. 37C strain3 target ssRNA 2 (SEQ. I.D. No. 301) FIG. 36A ssRNA 3 (SEQ. I.D. No.302) FIG. 36A

TABLE 11 plasmids used in this Example Plasmid Name Description Link toplasmid map pC004 beta-lactamase screening targethttps://benchling.com/s/lPJ1cCwR pC009 LshCas13a locus into pACYC184https://benchling.com/s/seqylkMuglYmiG4A3VhShZg with targeting spacerpC010 LshCas13a locus into pACYC184https://benchling.com/s/seq-2WApFr3zni1GOACyQY8a with nontargetingspacer pC011 LwCas13a locus into pACYC184https://benchling.com/s/seq-Vyk8qK2fyhzegfNgLJHM with targeting spacerpC012 LwCas13a locus into pACYC184https://benchling.com/s/seq-RxZAgPBzBUGQThkxR2Kx with nontargetingspacer pC013 Twinstrep-SUMO-huLwCas13a forhttps://benchling.com/s/seq-66CfLwu7sLMQMbcXe7Ih bacterial expression

Example 3—Characterization of Cas13b Orthologs with Orthogonal BasePreferences

Applicant biochemically characterized fourteen orthologs of the recentlydefined type VI CRISPR-Cas13b family of RNA-guided RNA-targeting enzymesto find new candidates for improving the SHERLOCK detection technology(FIGS. 83A and 85). Applicant was able to heterologously expressfourteen Cas13b orthologs in E. coli and purify the proteins for an invitro RNase activity assay (FIGS. 86A-86C). Because different Cas13orthologs might have varying base preferences for optimal cleavageactivity, Applicant generated fluorescent RNase homopolymer sensors thatconsisted of either 5 As, Gs, Cs, or Us to evaluate orthogonal cleavagepreferences. Applicant incubated each ortholog with its cognate crRNAtargeting a synthetic 173nt ssRNA 1 and measured collateral cleavageactivity using the homopolymer fluorescent sensors (FIGS. 83B and87A-87D).

Example 4—Motif Discovery Screen with Library

To further explore the diversity of cleavage preferences of the variousCas13a and Cas13b orthologs, Applicant developed a library-basedapproach for characterizing motifs preferred for endonuclease activityin response to collateral activity. Applicant used a degenerate 6-merRNA reporter flanked by constant DNA handles, which allowed foramplification and readout of uncleaved sequences (FIG. 83C). Incubatingthis library with collateral activated Cas13 enzymes resulted indetectable cleavage and depended on the addition of target RNA (FIG.88). Sequencing of depleted motifs revealed an increase in the skew ofthe library over digestion time (FIG. 89A), indicative ofbase-preference, and selecting sequences above a threshold ratioproduced number enriched sequences that corresponded with cleavage ofthe enzymes (FIG. 89B). Sequence logos from enriched motifs reproducedthe U-preference observed for LwaCas13a and CcaCas13b and theA-preference of PsmCas13b (FIGS. 89C-89E). Applicant also determinedmultiple sequences that showed cleavage for only one ortholog, but notothers, to allow for independent readout (FIG. 89F).

To understand the specific sub-motifs of enzyme preference, Applicantanalyzed the depleted motifs for single-base preferences (FIG. 90A),which agreed with homopolymer motifs tested as well as for two-basemotifs (FIGS. 83C and 90B). These two base motifs reveal a more complexpreference, especially for LwaCas13a and PsmCas13b, which prefers TA,GA, and AT dibase sequences. Higher order motifs also revealedadditional preferences (FIGS. 91 and 92A-92D).

Applicant confirmed the collateral preferences of LwaCas13a, PsmCas13b,and CcaCas13b with in vitro digestion of targets (FIGS. 93A-93C). Inorder to improve the weak digestion of PsmCas13b, Applicant optimizedthe buffer composition and enzyme concentration (FIG. 94A, 94B). Otherdications tested on PsmCas13b and Cas13b orthologs did not have largeeffects (FIGS. 95A-95F). Applicant also compared PsmCas13b to apreviously characterized A-preference Cas13 family member for two RNAtargets, and found comparable or improved sensitivity (FIG. 96A, 96B).From these results, Applicant compared kinetics of LwaCas13a andPsmCas13b, in separate reactions with independent reporters, and foundlow levels of cross-talk between the two channels (FIG. 83D).

Example 5—Single Molecule Detection with LwaCas13a, PsmCas13b, andCcaCas13b

A key feature of the SHERLOCK technology is that it enables singlemolecule detection (2 aM or 1 molecule/μL) by LwaCas13a collateral RNaseactivity. To characterize the sensitivity of Cas13b enzymes, Applicantperformed SHERLOCK with PsmCas13b and CcaCas13b, another highly activeCas13b enzyme with uridine preference (FIG. 83E). Applicant found thatLwaCas13a, PsmCas13b, and CcaCas13b were capable of achieving 2 aMdetection of two different RNA targets, ssRNA 1 and a synthetic ZikassRNA (FIGS. 83E, 97A, 97B, and 98). To investigate the robustness oftargeting with these three enzymes, Applicant designed eleven differentcrRNAs evenly spaced across ssRNA 1 and found that LwaCas13a mostconsistently achieved signal detection, while CcaCas13b and Psmcas13bboth showed much more variability in detection from crRNA to crRNA(FIGS. 99A and 99B). To identify the optimal crRNA for detection,Applicant varied the spacer length of PsmCas13b and CcaCas13b from 34-12nt and found that PsmCas13b had a peak sensitivity at a spacer length of30 while CcaCas13b had equivalent sensitivity above spacer lengths of28nt (FIGS. 100A and 100B). Applicant also tested if the detection limitcould be pushed beyond 2 aM, allowing for larger sample volume inputsinto SHERLOCK. By scaling up the pre-amplification RPA step, Applicantfound that both LwaCas13a and PsmCas13b could give significant detectionsignals for 200, 20, and 2zM input samples and allow for volume inputsof 250 μL and 540 μL.

Example 6—Quantitative SHERLOCK with RPA

As SHERLOCK relies on an exponential amplification, accuratequantitation of nucleic acids can be difficult. Applicant hypothesizedthat reducing the efficiency of the RPA step could improve thecorrelation between the input amount and the signal of the SHERLOCKreaction. Applicant observed that the kinetics of the SHERLOCK detectionwere very sensitive to primer concentration across a range of sampleconcentrations (FIGS. 101A-101D). Applicant diluted primerconcentrations, which increased both signal and quantitative accuracy(FIGS. 83G and 101E). This observation may be due to a decrease inprimer-dimer formation, allowing for more effective amplification whilepreventing saturation. Primer concentrations of 120 nM exhibited thegreatest correlation between signal and input (FIG. 101F). This accuracywas sustainable across a large range of concentrations down to theattomolar range (FIGS. 83H and 101G).

Example 7—Two Color Multiplexing with Orthogonal Cas13 Orthologs

An advantageous feature of nucleic acid diagnostics is the ability tosimultaneously detect multiple sample inputs, allowing for multiplexeddetection panels or for in sample controls. Orthogonal base preferencesof the Cas13 enzymes offer the opportunity to have multiplexed SHERLOCK.Applicant can assay the collateral activity of different Cas13 enzymesin the same reaction via fluorescent homopolymer sensors of differentbase identities and fluorophore colors, enabling multiple targets to besimultaneously measured (FIG. 84A). To demonstrate this concept,Applicant designed an LwaCas13a crRNA against the Zika virus ssRNA and aPsmCas13b crRNA against the Dengue virus ssRNA. Applicant found thatthis assay with both sets of Cas13-crRNA complexes in the same reaction,was capable of identifying if Zika or Dengue RNA, or both, were presentin the reaction (FIG. 84B). Applicant also found that because of theorthogonal preferences between CcaCas13b and PsmCas13b, that these twoenzymes could also be leveraged for multiplexed detection of Zika andDengue targets (FIGS. 102A-102C). Applicant was successfully able toextend this concept towards the entire SHERLOCK reaction, containingboth multiplexed RPA primers and Cas13-crRNA complexes. Applicantdesigned an LwaCas13a crRNA against P. aeruginosa and a PsmCas13b crRNAagainst S. aureus and were able to detect both DNA targets down to theattomolar range (FIG. 84C). Similarly, using both PsmCas13b andLwaCas13a Applicant was able to achieve attomolar multiplexed detectionof Zika and Dengue RNA using SHERLOCK (FIGS. 103A, 103B).

Applicant has shown that LwaCas13a enabled single nucleotide variantdetection and that this could be applied for rapid genotyping from humansaliva, but detection required two separate reactions: one for eachallele-sensing crRNA. To enable a single-reaction SHERLOCK genotyping,Applicant designed a LwaCas13a crRNA against the G-allele and aPsmCas13b crRNA against the A-allele of the rs601338 SNP, a variant inthe alpha(1,2)-fucosyltransferase FUT2 gene that associates withnorovirus resistance. Using this single-sample multiplexed approach,Applicant was able to successfully genotype four different humansubjects using their saliva and accurately identify whether they werehomozygous or heterozygous.

To further showcase the versatility of the Cas13 family of enzymes,Applicant simulated a therapeutic approach that involves Cas13 servingas both a companion diagnostic and the therapy itself. Applicantrecently developed PspCas13b for programmable RNA editing oftranscripts, which can be used for correction mutations in geneticdiseases, using a system called RNA Editing for Programmable A to IReplacement (REPAIR). Because diagnostics can be very useful when pairedwith therapies to guide treatment decisions or to monitor the outcome ofa treatment, Applicant thought that SHERLOCK could be used forgenotyping to guide the REPAIR treatment and also as a readout on theedited RNA to track the editing efficiency of the therapy (FIG. 84F).Applicant chose to demonstrate this theranostic concept to correct anAPC mutation (APC:c.1262G>A) in Familial adenomatous polyposis 1, aninherited disorder that involves cancer in the large intestine andrectum. Applicant designed healthy and mutant cDNAs of the APC gene andtransfected these into HEK293FT cells. Applicant was able to harvest theDNA from these cells and successfully genotype the correct samples usingsingle-sample multiplexed SHERLOCK with LwaCas13a and PsmCas13b (FIG.84G). Concurrently, Applicant designed and cloned guide RNAs for theREPAIR system and transfected cells that had the diseased genotype withthe guide RNA and dPspCas13b-ADAR2_(dd)(E488Q) REPAIR system. After 48hours, Applicant harvested RNA, which Applicant split for input intoSHERLOCK to detect the editing outcome and for next-generationsequencing (NGS) analysis to confirm the editing rate. Sequencingrevealed that Applicant achieved 43% editing with the REPAIR system(FIG. 84G) and was able to detect this with SHERLOCK as thehealthy-sensing crRNA showed higher signal than the non-targeting guidecontrol condition and the disease-sensing crRNA showed a decrease insignal (FIGS. 84H and 104A-104B). Overall the design and synthesis ofreagents for this assay took 3 days, the genotyping took 1 day, and thecorrection with REPAIR and sensing the editing rate took 3 days,yielding a total theranostics pipeline that lasts only 7 days.

Applicant has demonstrated the highly sensitive and specific detectionof nucleic acids using the type VI RNA-guided RNA-targetingCRISPR-Cas13a ortholog from Leptotrichia wadei. Applicant has furthershown that the Cas13b family of enzymes are active biochemically andhave unique properties that make them amenable for multiplexed detectionof nucleic acids by SHERLOCK. By characterizing the orthogonal basepreferences of the Cas13b enzymes, Applicant found specific sequences offluorescent RNA sensors that are recognized by PsmCas13b that LwaCas13adoes not recognize. Applicant was able to leverage these basepreferences to make in-sample multiplexed detection of two differenttargets possible and show the utility of this feature for distinguishingviral strains and genotyping individuals. Additionally, throughengineering of the pre-amplification step, SHERLOCK can be madequantitative, allowing for approximation of the input nucleic acidconcentration or quantitation. Applicant has additionally shown that theorthogonal PsmCas13b is capable of single molecule detection and thatthrough scaling up the volume Applicant can perform detection of samplesup to ˜0.5 mL and down to concentrations of 2zM.

Multiplexed detection with SHERLOCK is possible by spatially performingmultiple reactions, but in-sample multiplexing via orthogonal basepreferences allows for many targets to be detected at scale and forcheaper cost. While Applicant has shown here two-input multiplexing, thecleavage motif screens enable the design of additional orthogonalcleavage sensors (FIGS. 90A-90C). LwaCas13a and CcaCas13b, which bothcleave the same uridine homopolymer and are thus not orthogonal asmeasured by homopolymer sensors (FIG. 83B), showed very unique cleavagepreferences by the motif screens (FIG. 90). By screening additionalCas13a, Cas13b, and Cas13c orthologs, it is likely that many orthologswill reveal unique 6-mer motif preferences, which could theoreticallyallow for highly-multiplexed SHERLOCK limited only by the number ofspectrally-unique fluorescent sensors. Highly-multiplexed SHERLOCKenables many technological applications, especially those involvingcomplex input sensing and logical computation.

These additional refinements of Cas13-based detection for visual, moresensitive, and multiplexed readouts enable increased applications fornucleic acid detection, especially in settings where portable andinstrument-free analysis are necessary. Rapid multiplexed genotyping caninform pharmacogenomic decisions, test for multiple crop traits in thefield, or assess for the presence of co-occurring pathogens. Rapid,isothermal readout increases the accessibility of this detection forsettings where power or portable readers are unavailable, even for rarespecies like circulating DNA. Improved CRISPR-based nucleic acid testsmake it easier to understand the presence of nucleic acids inagriculture, pathogen detection, and chronic diseases.

Further embodiments of the invention are described in the followingnumbered paragraphs.

1. A nucleic acid detection system comprising:

a detection CRISPR system comprising an effector protein and one or moreguide RNAs designed to bind to corresponding target molecules; and

an RNA-based masking construct.

2. A polypeptide detection system comprising:

a detection CRISPR system comprising an effector protein and one or moreguide RNAs designed to bind to a trigger RNA;

an RNA-based masking construct; and

one or more detection aptamers comprising a masked RNA polymerasepromoter binding site or a masked primer binding site.

3. The system of paragraphs 1 or 2, further comprising nucleic acidamplification reagents.

4. The system of paragraph 1, wherein the target molecule is a targetDNA and the system further comprises a primer that binds the target DNAand comprises an RNA polymerase promoter.

5. The system of any one of paragraphs 1 to 4, wherein the CRISPR systemeffector protein is an RNA-targeting effector protein.

6. The system of paragraph 5, wherein the RNA-targeting effector proteincomprises one or more HEPN domains.

7. The system of paragraph 6, wherein the one or more HEPN domainscomprise a RxxxxH motif sequence.

8. The system of paragraph 7, wherein the RxxxH motif comprises aR{N/H/K]X₁X₂X₃H sequence.

9. The system of paragraph 8, wherein X₁ is R, S, D, E, Q, N, G, or Y,and X2 is independently I, S, T, V, or L, and X3 is independently L, F,N, Y, V, I, S, D, E, or A.

10. The system of any one of paragraphs 1 to 9, wherein the CRISPRRNA-targeting effector protein is C2c2.

11. The system of paragraph 6, wherein the CRISPR RNA-targeting effectorprotein is C2c2.

12. The system of paragraph 11, wherein the C2c2 is within 20 kb of aCas 1 gene.

13. The system of paragraph 12, wherein the C2c2 effector protein isfrom an organism of a genus selected from the group consisting of:Leptotrichia, Listeria, Corynebacter, Sutterella, Legionella, Treponema,Filifactor, Eubacterium, Streptococcus, Lactobacillus, Mycoplasma,Bacteroides, Flaviivola, Flavobacterium, Sphaerochaeta, Azospirillum,Gluconacetobacter, Neisseria, Roseburia, Parvibaculum, Staphylococcus,Nitratifractor, Mycoplasma, Campylobacter, and Lachnospira.

14. The system of paragraph 13, wherein the C2c2 or Cas13b effectorprotein is from an organism selected from the group consisting of:Leptotrichia shahii; Leptotrichia wadei (Lw2); Listeria seeligeri;Lachnospiraceae bacterium MA2020; Lachnospiraceae bacterium NK4A 179;[Clostridium] aminophilum DSM 10710; Carnobacterium gallinarum DSM 4847;Carnobacterium gallinarum DSM 4847 (second CRISPR Loci); Paludibacterpropionicigenes WB4; Listeria weihenstephanensis FSL R9-0317;Listeriaceae bacterium FSL M6-0635; Leptotrichia wadei F0279;Rhodobacter capsulatus SB 1003; Rhodobacter capsulatus R121; Rhodobactercapsulatus DE442; Leptotrichia buccalis C-1013-b; Herbinixhemicellulosilytica; [Eubacterium] rectale; Eubacteriaceae bacteriumCHKCI004; Blautia sp. Marseille-P2398; Leptotrichia sp. oral taxon 879str. F0557; Lachnospiraceae bacterium NK4A 144; Chloroflexus aggregans;Demequina aurantiaca; Thalassospira sp. TSL5-1; Pseudobutyrivibrio sp.OR37; Butyrivibrio sp. YAB3001; Blautia sp. Marseille-P2398;Leptotrichia sp. Marseille-P 3007; Bacteroides ihuae; Porphyromonadaceaebacterium KH3CP3RA; Listeria riparia; and Insolitispirillum peregrinum.

15. The system of paragraph 14, wherein the C2c2 effector protein is aL. wadei F0279 or L. wadei F0279 (Lw2) C2c2 effector protein.

16. The system of any one of paragraphs 1 to 15, wherein the RNA-basedmasking construct suppresses generation of a detectable positive signal.

17. The system of paragraph 16, wherein the RNA-based masking constructsuppresses generation of a detectable positive signal by masking thedetectable positive signal, or generating a detectable negative signalinstead.

18. The system of paragraph 16, wherein the RNA-based masking constructcomprises a silencing RNA that suppresses generation of a gene productencoded by a reporting construct, wherein the gene product generates thedetectable positive signal when expressed.

19. The system of paragraph 16, wherein the RNA-based masking constructis a ribozyme that generates the negative detectable signal, and whereinthe positive detectable signal is generated when the ribozyme isdeactivated.

20. The system of paragraph 19, wherein the ribozyme converts asubstrate to a first color and wherein the substrate converts to asecond color when the ribozyme is deactivated.

21. The system of paragraph 16, wherein the RNA-based masking agent isan RNA aptamer and/or comprises an RNA-tethered inhibitor.

22. The system of paragraph 21, wherein the aptamer or RNA-tetheredinhibitor sequesters an enzyme, wherein the enzyme generates adetectable signal upon release from the aptamer or RNA tetheredinhibitor by acting upon a substrate.

23. The system of paragraph 21, wherein the aptamer is an inhibitoryaptamer that inhibits an enzyme and prevents the enzyme from catalyzinggeneration of a detectable signal from a substrate or wherein theRNA-tethered inhibitor inhibits an enzyme and prevents the enzyme fromcatalyzing generation of a detectable signal from a substrate.

24. The system of paragraph 23, wherein the enzyme is thrombin, proteinC, neutrophil elastase, subtilisin, horseradish peroxidase,beta-galactosidase, or calf alkaline phosphatase.

25. The system of paragraph 24, wherein the enzyme is thrombin and thesubstrate is para-nitroanilide covalently linked to a peptide substratefor thrombin, or 7-amino-4-methylcoumarin covalently linked to a peptidesubstrate for thrombin.

26. The system of paragraph 21, wherein the aptamer sequesters a pair ofagents that when released from the aptamers combine to generate adetectable signal.

27. The system of paragraph 16, wherein the RNA-based masking constructcomprises an RNA oligonucleotide to which a detectable ligand and amasking component are attached.

28. The system of paragraph 16, wherein the RNA-based masking constructcomprises a nanoparticle held in aggregate by bridge molecules, whereinat least a portion of the bridge molecules comprises RNA, and whereinthe solution undergoes a color shift when the nanoparticle is disbursedin solution.

29. The system of paragraph 28, wherein the nanoparticle is a colloidalmetal.

30. The system of paragraph 29, wherein the colloidal metal is colloidalgold.

31. The system of paragraph 16, wherein the RNA-based masking constructcomprising a quantum dot linked to one or more quencher molecules by alinking molecule, wherein at least a portion of the linking moleculecomprises RNA.

32. The system of paragraph 16, wherein the RNA-based masking constructcomprises RNA in complex with an intercalating agent, wherein theintercalating agent changes absorbance upon cleavage of the RNA.

33. The system of paragraph 32, wherein the intercalating agent ispyronine-Y or methylene blue.

34. The system of paragraph 16, wherein the detectable ligand is afluorophore and the masking component is a quencher molecule.

34. The system according to any of paragraphs 1 to 34, wherein the oneor more guide RNAs designed to bind to corresponding target moleculescomprise a (synthetic) mismatch.

35. The system according to paragraph 34, wherein said mismatch is up-or downstream of a SNP or other single nucleotide variation in saidtarget molecule.

36. The system of any one of paragraphs 1 to 35, wherein the one or moreguide RNAs are designed to detect a single nucleotide polymorphism in atarget RNA or DNA, or a splice variant of an RNA transcript.

37. The system of any one of paragraphs 1 to 36, wherein the one or moreguide RNAs are designed to bind to one or more target molecules that arediagnostic for a disease state.

38. The system of paragraph 37, wherein the disease state is cancer.

39. The system of paragraph 38, wherein the disease state is anautoimmune disease.

40. The system of paragraph 37, wherein the disease state is aninfection.

41. The system of paragraph 40, wherein the infection is caused by avirus, a bacterium, a fungus, a protozoa, or a parasite.

42. The system of paragraph 41, wherein the infection is a viralinfection.

43. The system of paragraph 42, wherein the viral infection is caused bya DNA virus.

44. The system of paragraph 43, wherein the DNA virus is a Myoviridae,Podoviridae, Siphoviridae, Alloherpesviridae, Herpesviridae (includinghuman herpes virus, and Varicella Zoster virus), Malocoherpesviridae,Lipothrixviridae, Rudiviridae, Adenoviridae, Ampullaviridae,Ascoviridae, Asfarviridae (including African swine fever virus),Baculoviridae, Cicaudaviridae, Clavaviridae, Corticoviridae,Fuselloviridae, Globuloviridae, Guttaviridae, Hytrosaviridae,Iridoviridae, Maseilleviridae, Mimiviridae, Nudiviridae, Nimaviridae,Pandoraviridae, Papillomaviridae, Phycodnaviridae, Plasmaviridae,Polydnaviruses, Polyomaviridae (including Simian virus 40, JC virus, BKvirus), Poxviridae (including Cowpox and smallpox), Sphaerolipoviridae,Tectiviridae, Turriviridae, Dinodnavirus, Salterprovirus, Rhizidovirus.

45. The system of paragraph 42, wherein the viral infection is caused bya double-stranded RNA virus, a positive sense RNA virus, a negativesense RNA virus, a retrovirus, or a combination thereof.

46. The system of paragraph 45, wherein the viral infection is caused bya Coronaviridae virus, a Picornaviridae virus, a Caliciviridae virus, aFlaviviridae virus, a Togaviridae virus, a Bornaviridae, a Filoviridae,a Paramyxoviridae, a Pneumoviridae, a Rhabdoviridae, an Arenaviridae, aBunyaviridae, an Orthomyxoviridae, or a Deltavirus.

47. The system of paragraph 46, wherein the viral infection is caused byCoronavirus, SARS, Poliovirus, Rhinovirus, Hepatitis A, Norwalk virus,Yellow fever virus, West Nile virus, Hepatitis C virus, Dengue fevervirus, Zika virus, Rubella virus, Ross River virus, Sindbis virus,Chikungunya virus, Borna disease virus, Ebola virus, Marburg virus,Measles virus, Mumps virus, Nipah virus, Hendra virus, Newcastle diseasevirus, Human respiratory syncytial virus, Rabies virus, Lassa virus,Hantavirus, Crimean-Congo hemorrhagic fever virus, Influenza, orHepatitis D virus.

48. The system of paragraph 41, wherein the infection is a bacterialinfection.

49. The system of paragraph 48, wherein the bacterium causing thebacterial infection is Acinetobacter species, Actinobacillus species,Actinomycetes species, an Actinomyces species, Aerococcus species anAeromonas species, an Anaplasma species, an Alcaligenes species, aBacillus species, a Bacteroides species, a Bartonella species, aBifidobacterium species, a Bordetella species, a Borrelia species, aBrucella species, a Burkholderia species, a Campylobacter species, aCapnocytophaga species, a Chlamydia species, a Citrobacter species, aCoxiella species, a Corynbacterium species, a Clostridium species, anEikenella species, an Enterobacter species, an Escherichia species, anEnterococcus species, an Ehlichia species, an Epidermophyton species, anErysipelothrix species, a Eubacterium species, a Francisella species, aFusobacterium species, a Gardnerella species, a Gemella species, aHaemophilus species, a Helicobacter species, a Kingella species, aKlebsiella species, a Lactobacillus species, a Lactococcus species, aListeria species, a Leptospira species, a Legionella species, aLeptospira species, Leuconostoc species, a Mannheimia species, aMicrosporum species, a Micrococcus species, a Moraxella species, aMorganell species, a Mobiluncus species, a Micrococcus species,Mycobacterium species, a Mycoplasm species, a Nocardia species, aNeisseria species, a Pasteurelaa species, a Pediococcus species, aPeptostreptococcus species, a Pityrosporum species, a Plesiomonasspecies, a Prevotella species, a Porphyromonas species, a Proteusspecies, a Providencia species, a Pseudomonas species, aPropionibacteriums species, a Rhodococcus species, a Rickettsia species,a Rhodococcus species, a Serratia species, a Stenotrophomonas species, aSalmonella species, a Serratia species, a Shigella species, aStaphylococcus species, a Streptococcus species, a Spirillum species, aStreptobacillus species, a Treponema species, a Tropheryma species, aTrichophyton species, an Ureaplasma species, a Veillonella species, aVibrio species, a Yersinia species, a Xanthomonas species, orcombination thereof.

50. The system of paragraph 41, wherein the infection is caused by afungus.

51. The system of paragraph 50, wherein the fungus is Aspergillus,Blastomyces, Candidiasis, Coccidiodomycosis, Cryptococcus neoformans,Cryptococcus gatti, sp. Histoplasma sp. (such as Histoplasmacapsulatum), Pneumocystis sp. (such as Pneumocystis jirovecii),Stachybotrys (such as Stachybotrys chartarum), Mucroymcosis, Sporothrix,fungal eye infections ringworm, Exserohilum, Cladosporium, Geotrichum,Saccharomyces, a Hansenula species, a Candida species, a Kluyveromycesspecies, a Debaryomyces species, a Pichia species, a Penicilliumspecies, a Cladosporium species, a Byssochlamys species or a combinationthereof.

52. The system of paragraph 41, wherein the infection is caused by aprotozoa.

53. The system of paragraph 52, wherein the protozoa is Euglenozoa, aHeterolobosea, a Diplomonadida, an Amoebozoa, a Blastocystic, anApicomplexa, or combination thereof.

54. The system of paragraph 41, wherein the infection is caused by aparasite.

55. The system of paragraph 54, wherein the parasite is Trypanosomacruzi (Chagas disease), T. brucei gambiense, T. brucei rhodesiense,Leishmania braziliensis, L. infantum, L. mexicana, L. major, L. tropica,L. donovani, Naegleria fowleri, Giardia intestinalis (G. lamblia, G.duodenalis), canthamoeba castellanii, Balamuthia madrillaris, Entamoebahistolytica, Blastocystic hominis, Babesia microti, Cryptosporidiumparvum, Cyclospora cayetanensis, Plasmodium falciparum, P. vivax, P.ovale, P. malariae, and Toxoplasma gondii, or combination thereof.

56. The system of any one of paragraphs 1 to 55, wherein the reagents toamplify target RNA molecules comprise nucleic acid sequence-basedamplification (NASBA), recombinase polymerase amplification (RPA),loop-mediated isothermal amplification (LAMP), strand displacementamplification (SDA), helicase-dependent amplification (HDA), nickingenzyme amplification reaction (NEAR), PCR, multiple displacementamplification (MDA), rolling circle amplification (RCA), ligase chainreaction (LCR), or ramification amplification method (RAM).

57. The system of any one of paragraphs 1 to 56, further comprising anenrichment CRISPR system, wherein the enrichment CRISPR system isdesigned to bind the corresponding target molecules prior to detectionby the detection CRISPR system.

58. The system of paragraph 57, wherein the enrichment CRISPR systemcomprises a catalytically inactive CRISPR effector protein.

59. The system of paragraph 58, wherein catalytically inactive CRISPReffector protein is a catalyically inactive C2c2.

60. The system of any one of paragraphs 57 to 59, wherein the enrichmentCRISPR effector protein further comprises a tag, wherein the tag is usedto pull down the enrichment CRISPR effector system, or to bind theenrichment CRISPR system to a solid substrate.

61. The system of paragraph 60, wherein the solid substrate is a flowcell.

62. A diagnostic device comprising one or more individual discretevolumes, each individual discrete volume comprising a CRISPR system ofany one of paragraphs 1 to 61.

63. The diagnostic device of paragraph 62, wherein each individualdiscrete volume further comprises one or more detection aptamerscomprising a masked RNA polymerase promoter binding site or a maskedprimer binding site.

64. The device of paragraphs 62 or 63, wherein each individual discretevolume further comprises nucleic acid amplification reagents.

65. The device of paragraph 62, wherein the target molecule is a targetDNA and the individual discrete volumes further comprise a primer thatbinds the target DNA and comprises an RNA polymerase promoter.

66. The device of any one of paragraphs 62 to 65, wherein the individualdiscrete volumes are droplets.

67. The device of any one of paragraphs 62 to 66, wherein the individualdiscrete volumes are defined on a solid substrate.

68. The device of paragraph 67, wherein the individual discrete volumesare microwells.

69. The diagnostic device of any one of paragraphs 62 to 66, wherein theindividual discrete volumes are spots defined on a substrate.

70. The device of paragraph 69, wherein the substrate is a flexiblematerials substrate.

71. The device of paragraph 70, wherein the flexible materials substrateis a paper substrate or a flexible polymer based substrate.

72. A method for detecting target nucleic acids in samples, comprising:

distributing a sample or set of samples into one or more individualdiscrete volumes, the individual discrete volumes comprising a CRISPRsystem of any one of paragraphs 1 or 3 to 61;

incubating the sample or set of samples under conditions sufficient toallow binding of the one or more guide RNAs to one or more targetmolecules;

activating the CRISPR effector protein via binding of the one or moreguide RNAs to the one or more target molecules, wherein activating theCRISPR effector protein results in modification of the RNA-based maskingconstruct such that a detectable positive signal is generated; and

detecting the detectable positive signal, wherein detection of thedetectable positive signal indicates a presence of one or more targetmolecules in the sample.

73. A method for detecting polypeptides in samples, comprising:

distributing a sample or set of samples into a set of individualdiscrete volumes, the individual discrete volumes comprising peptidedetection aptamers, a CRISPR system of any one of paragraphs 2 to 61;

incubating the sample or set of samples under conditions sufficient toallow binding of the peptide detection aptamers to the one or moretarget molecules, wherein binding of the aptamer to a correspondingtarget molecule exposes the RNA polymerase binding site or primerbinding site resulting in generation of a trigger RNA;

activating the RNA effector protein via binding of the one or more guideRNAs to the trigger RNA, wherein activating the RNA effector proteinresults in modification of the RNA-based masking construct such that adetectable positive signal is produced; and

detecting the detectable positive signal, wherein detection of thedetectable positive signal indicates a presence of one or more targetmolecules in a sample.

74. The method of paragraph 72, wherein the target molecule is a targetDNA and the method further comprising binding the target DNA with aprimer comprising an RNA polymerase site.

75. The method of any one of paragraphs 72 to 74, further comprisingamplifying the sample RNA or the trigger RNA.

76. The method of paragraph 75, wherein amplifying RNA comprisesamplification by NASBA.

77. The method of paragraph 75, wherein amplifying RNA comprisesamplification by RPA.

78. The method of any one of paragraphs 72 to 77, wherein the sample isa biological sample or an environmental sample.

79. The method of paragraph 78, wherein biological sample is a blood,plasma, serum, urine, stool, sputum, mucous, lymph fluid, synovialfluid, bile, ascites, pleural effusion, seroma, saliva, cerebrospinalfluid, aqueous or vitreous humor, or any bodily secretion, a transudate,an exudate (for example, fluid obtained from an abscess or any othersite of infection or inflammation), or fluid obtained from a joint (forexample, a normal joint or a joint affected by disease, such asrheumatoid arthritis, osteoarthritis, gout or septic arthritis), or aswab of skin or mucosal membrane surface.

80. The method of paragraph 78, wherein the environmental sample isobtained from a food sample, paper surface, a fabric, a metal surface, awood surface, a plastic surface, a soil sample, a fresh water sample, awaste water sample, a saline water sample, or a combination thereof.

81. The method of any one of paragraphs 72 or 74 to 80, wherein the oneor more guide RNAs are designed to detect a single nucleotidepolymorphism in a target RNA or DNA, or a splice variant of an RNAtranscript.

82. The method of any one of paragraphs 72 to 81, wherein the one ormore guide RNAs are designed to bind to one or more target moleculesthat are diagnostic for a disease state.

83. The method of any one of paragraphs 81 to 82, wherein the one ormore guide RNAs are designed to bind to cell free nucleic acids.

84. The method of paragraph 82, wherein the disease state is aninfection, an organ disease, a blood disease, an immune system disease,a cancer, a brain and nervous system disease, an endocrine disease, apregnancy or childbirth-related disease, an inherited disease, or anenvironmentally-acquired disease.

85. The system of paragraph 37, wherein said disease state ischaracterized by the presence or absence of an antibiotic or drugresistance or susceptibility gene or transcript or polypeptide,preferably in a pathogen or a cell.

86. The system of paragraph 37, wherein said target molecule is anantibiotic or drug resistance or susceptibility gene or transcript orpolypeptide.

87. The system of paragraph 35, wherein the synthetic mismatch in saidguide RNA is at position 3, 4, 5, or 6 of the spacer, preferablyposition 3.

88. The system of paragraph 34, 35, or 82, wherein said mismatch in saidguide RNA is at position 1, 2, 3, 4, 5, 6, 7, 8, or 9 of the spacer,preferably position 5.

89. The system of paragraph 35 or 82, wherein said mismatch is 1, 2, 3,4, or 5 nucleotides upstream or downstream, preferably 2 nucleotides,preferably downstream of said SNP or other single nucleotide variationin said guide RNA.

90. The system of any of paragraphs 1-56 or 89, wherein said guide RNAcomprises a spacer which is truncated relative to a wild type spacer.

91. The system of any of paragraphs 1-56 or 80-85, wherein said guideRNA comprises a spacer which comprises less than 28 nucleotides,preferably between and including 20 to 27 nucleotides.

92. The system of any of paragraphs 1-56 or 80-85, wherein said guideRNA comprises a spacer which consists of 20-25 nucleotides or 20-23nucleotides, such as preferably 20 or 23 nucleotides.

93. The system of any of paragraphs 1-56 or 85-92, wherein said maskingconstruct comprises an RNA oligonucleotide designed to bind aG-quadruplex forming sequence, wherein a G-quadruplex structure isformed by the G-quadruplex forming sequence upon cleavage of the maskingconstruct, and wherein the G-quadruplex structure generates a detectablepositive signal.

94. The method of any of paragraphs 72 to 84, further comprisingcomparing the detectable positive signal with a (synthetic) standardsignal.

95. A method for detecting a target nucleic acid in a sample,comprising:

contacting a sample with a nucleic acid detection system according toany of paragraphs 1 to 56; and

applying said contacted sample to a lateral flow immunochromatographicassay.

96. The method according to paragraph 94, wherein said nucleic aciddetection system comprises an RNA-based masking construct comprising afirst and a second molecule, and wherein said lateral flowimmunochromatographic assay comprises detecting said first and secondmolecule, preferably at discrete detection sites on a lateral flowstrip.

97. The method according to paragraph 95, wherein said first moleculeand said second molecule is detected by binding to an antibodyrecognizing said first or second molecule and detecting said boundmolecule, preferably with sandwich antibodies.

98. The method according to paragraph 94 or 95, wherein said lateralflow strip comprises an upstream first antibody directed against saidfirst molecule, and a downstream second antibody directed against saidsecond molecule, and wherein uncleaved RNA-based masking construct isbound by said first antibody if the target nucleic acid is not presentin said sample, and wherein cleaved RNA-based masking construct is boundboth by said first antibody and said second antibody if the targetnucleic acid is present in said sample.

Various modifications and variations of the described methods,pharmaceutical compositions, and kits of the invention will be apparentto those skilled in the art without departing from the scope and spiritof the invention. Although the invention has been described inconnection with specific embodiments, it will be understood that it iscapable of further modifications and that the invention as claimedshould not be unduly limited to such specific embodiments. Indeed,various modifications of the described modes for carrying out theinvention that are obvious to those skilled in the art are intended tobe within the scope of the invention. This application is intended tocover any variations, uses, or adaptations of the invention following,in general, the principles of the invention and including suchdepartures from the present disclosure come within known customarypractice within the art to which the invention pertains and may beapplied to the essential features herein before set forth.

What is claimed is:
 1. A nucleic acid detection system comprising: adetection CRISPR system comprising an effector protein and one or moreguide RNAs designed to bind to corresponding target molecules; and anRNA-based masking construct.
 2. A polypeptide detection systemcomprising: a detection CRISPR system comprising an effector protein andone or more guide RNAs designed to bind to a trigger RNA; an RNA-basedmasking construct; and one or more detection aptamers comprising amasked RNA polymerase promoter binding site or a masked primer bindingsite.
 3. The system of claim 1 or 2, further comprising nucleic acidamplification reagents.
 4. The system of claim 1, wherein the targetmolecule is a target DNA and the system further comprises a primer thatbinds the target DNA and comprises an RNA polymerase promoter.